Anti-pd-1 antibodies and their uses

ABSTRACT

The present disclosure provides novel anti-PD-1 antibodies, compositions including the new antibodies, nucleic acids encoding the antibodies, and methods of making and using the same.

1. CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 62/394,314, filed Sep. 14, 2016, thecontents of which are incorporated herein in its entirety by referencethereto.

2. SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 10, 2017, isnamed 381493-327US_SL.txt and is 32,733 bytes in size.

3. TECHNICAL FIELD

The present application pertains to, among other things, novel anti-PD-1antibodies, compositions including the new antibodies, nucleic acidsencoding the antibodies, and methods of making and using the same.

4. BACKGROUND

Cancer therapies comprise a wide range of therapeutic approaches,including surgery, radiation, and chemotherapy. While the variousapproaches allow a broad selection of treatments to be available to themedical practitioner to treat the cancer, existing therapeutics sufferfrom a number of disadvantages, such as a lack of selectivity oftargeting cancer cells over normal, healthy cells, and the developmentof resistance by the cancer to the treatment.

Recent approaches based on targeted therapeutics, which interfere withcellular processes of cancer cells preferentially over normal cells,have led to chemotherapeutic regimens with fewer side effects ascompared to non-targeted therapies such as radiation treatment.

Cancer immunotherapy has emerged as a promising therapeutic approach tocomplement existing standards of care. See, e.g., Miller, et al. CancerCell, 27, 439-449 (2015). Such immunotherapy approaches include thedevelopment of antibodies used to modulate the immune system to killcancer cells.

For example, interaction of PD-1, a type I cell surface receptor, witheither of its two ligands, PD-L1 or PD-L2, results in a dominantnegative checkpoint signal that limits subsequent antigenreceptor-driven cellular activation. The ligands for PD-1 aredifferentially expressed on various tissues and cell types, includingantigen-presenting cells of the immune system, and are upregulated onmany types of tumor cells. Upregulation of PD-L1 within the tumormicroenvironment is a proposed mechanism of tumors to subvert protectiveanti-tumor immune responses by the host. Antibodies directed at PD-1that block the interaction of the receptor with either of its ligandsresult in inhibition of negative signaling. In vitro inhibition of thePD-1 mediated checkpoint signal has been demonstrated to result inprolonged antigen-specific T cell activation. In vivo PD-1 blockade hasbeen shown to enhance anti-tumor immune responses in both syngeneicmouse tumor models and in human clinical trials.

Anti-tumor immune responses in patients with solid tumors have beenenhanced by anti-PD-1 treatment. There are two approved and marketedantagonistic anti-PD-1 monoclonal antibodies: nivolumab (OPDIVO®) andpembrolizumab (KEYTRUDA®), with approvals in the US and the EuropeanUnion to treat diseases such as unresectable or metastatic melanoma andmetastatic non-small cell lung cancer. Treatment of patients with theseagents has resulted in anti-tumor responses as measured by improvementin either progression free survival and/or overall survival.

The recent failure of OPDIVO® to slow progression of advanced lungcancer in a treatment-naïve patient population in a clinical trialcomparing OPDIVO® with conventional chemotherapy highlights the need foralternative approaches and additional cancer treatments to complementexisting therapeutic standards of care.

5. SUMMARY

The present disclosure provides anti-PD-1 antibodies and bindingfragments thereof that specifically bind to PD-1. The amino acidsequences of exemplary CDRs, as well as the amino acid sequence of theV_(H) and V_(L) regions of the heavy and light chains of exemplaryanti-PD-1 antibodies are provided in the Detailed Description below.Antibodies provided herein interfere with the interaction of the PD-1receptor with either of its ligands (PD-L1, SEQ ID NO:3; PD-L2, SEQ IDNO:4), resulting in inhibition of negative signaling and upregulation ofthe adaptive immune response.

The anti-PD-1 antibodies may include modifications and/or mutations thatalter the properties of the antibodies, such as increase half-life,increase or decrease ADCC, etc., as is known in the art.

Nucleic acids comprising nucleotide sequences encoding the anti-PD-1antibodies of the disclosure are provided herein, as are vectorscomprising nucleic acids. Additionally, prokaryotic and eukaryotic hostcells transformed with a vector comprising a nucleotide sequenceencoding a disclosed anti-PD-1 antibody are provided herein, as well aseukaryotic (such as mammalian) host cells engineered to express thenucleotide sequences. Methods of producing antibodies, by culturing hostcells and recovering the antibodies are also provided, and discussedfurther in the Detailed Description below.

In another aspect, the present disclosure provides compositionsincluding the anti-PD-1 antibodies described herein. The compositionsgenerally comprise one or more anti-PD-1 antibody as described herein,and/or salts thereof, and one or more excipients, carriers or diluents.

The present disclosure provides methods of treating subjects, such ashuman subjects, diagnosed with a solid tumor or a blood malignancy withan anti-PD-1 antibody. The method generally involves administering tothe subject an amount of an anti-PD-1 antibody described hereineffective to provide therapeutic benefit. The subject may be diagnosedwith any one of a number of solid tumors or blood malignancies that maybe newly diagnosed, relapsed, or relapsed and refractory. An anti-PD-1antibody is typically administered as an intravenous infusion twice aweek, once a week, once every two weeks, once every three weeks, onceevery four weeks, once every five weeks, once every six weeks, onceevery seven weeks, or once every eight weeks.

The anti-PD-1 antibodies may be administered as single therapeuticagents (monotherapy) or adjunctive to or with other therapeutic agentstypically, but not necessarily, those used for the treatment of a solidtumor or blood malignancy. Therapeutic agents typically will be used attheir approved dose, route of administration, and frequency ofadministration, but may be used at lower dosages.

The anti-PD-1 antibodies may be administered via a variety of routes ormodes of administration, including but not limited to, intravenousinfusion and/or injection, intratumoral injection, and subcutaneousinjection. The amount administered will depend upon the route ofadministration, the dosing schedule, the type of cancer being treated,the stage of the cancer being treated, and other parameters such as theage and weight of the patient, as is well known in the art. Specificexemplary dosing schedules expected to provide therapeutic benefit areprovided in the Detailed Description.

6. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B show the amino acid sequences of human PD-1 (SEQ ID NO:1),murine PD-1 (SEQ ID NO:2), human PD-L1 (SEQ ID NO:3), and human PD-L2(SEQ ID NO:4). FIG. 1A depicts the sequences of human and murine PD-1;FIG. 1B depicts the sequences of human PD-L1 and PD-L2.

FIG. 2 provides amino acid sequences of V_(H) and V_(L) regions inexemplary anti-PD-1 antibodies of the disclosure.

FIGS. 3A-3C show the effects of oxidative and variable temperatureconditions on exemplary humanized antibody Hu12A11.2b1 and its pointmutations at Kabat 99 in CDR-H3. FIG. 3A depicts binding to PD-1 inJurkat cells of Hu12A11.2b1 stored at −80, 5, 25, or 40° C. for 30 daysor after being subjected to oxidative conditions (1% hydrogen peroxide,“1% HP”; or 1% tert-butyl hydroperoxide, “1% TBHP”); FIG. 3B depictsbinding of anti-PD-1 antibodies Hu12A11.2b1, Hu12A11.2b2, Hu12A11.2b3,and Hu12A11.2b4; FIG. 3C depicts the binding of Hu12A11.2b4 to PD-1 inJurkat cells after incubation at −80, 5, 25, or 40° C. for 30 days.

FIGS. 4A-4B show the biological activity of nivolumab and Hu12A11.2b4 inmixed leukocyte response (MLR) and tetanus toxoid antigen recall assays.FIG. 4A shows enhancement in IL-2 or interferon-gamma (IFN-γ) levelsafter treatment with 10 μg/mL of nivolumab, Hu12A11.2b4, or isotypecontrol in mixed leukocyte cultures. FIG. 4B shows dose-dependentresponse of nivolumab, Hu12A11.2b4, or isotype control on IFN-γenhancement in tetanus toxoid response assay.

7. DETAILED DESCRIPTION

The present disclosure concerns antibodies and fragments thatspecifically bind PD-1, compositions comprising the antibodies,polynucleotides encoding anti-PD-1 antibodies, host cells capable ofproducing the antibodies, methods and compositions useful for making theantibodies and binding fragments, and various methods of using the same.

As will be appreciated by skilled artisans, antibodies are “modular” innature. Throughout the disclosure, various specific embodiments of thevarious “modules” composing the antibodies are described. As specificnon-limiting examples, various specific embodiments of V_(H) CDRs, V_(H)chains, V_(L) CDRs and V_(L) chains are described. It is intended thatall of the specific embodiments may be combined with each other asthough each specific combination were explicitly described individually.

7.1. Abbreviations

The antibodies, binding fragments, and polynucleotides described hereinare, in many embodiments, described by way of their respectivepolypeptide or polynucleotide sequences. Unless indicated otherwise,polypeptide sequences are provided in N→C orientation; polynucleotidesequences in 5′→3′ orientation. For polypeptide sequences, theconventional three or one-letter abbreviations for the geneticallyencoded amino acids may be used, as noted in TABLE 1, below.

TABLE 1 Encoded Amino Acid Abbreviations Amino Acid Three LetterAbbreviation One-Letter Abbreviation Alanine Ala A Arginine Arg RAsparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamic acid Glu EGlutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I LeucineLeu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro PSerine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine ValV

Certain sequences are defined by structural formulae specifying aminoacid residues belonging to certain classes (e.g., aliphatic,hydrophobic, etc.). The various classes to which the genetically encodedamino acids belong as used herein are noted in TABLE 2, below. Someamino acids may belong to more than one class. Cysteine, which containsa sulfhydryl group, and proline, which is conformationally constrained,are not assigned classes.

TABLE 2 Encoded Amino Acid Classes Class Amino Acids Aliphatic A, I, L,VAromatic F, Y, W Non-Polar M, A, I, L, V Polar N, Q, S, T Basic H, K, RAcidic D, E Small A, G

The abbreviations used for the various exemplary antibodies disclosedherein are provided in TABLE 3, below:

TABLE 3 Antibody Abbreviations Clone/Name Abbreviation V_(H) Sequence(FIG. 2) V_(L) Sequence (FIG. 2) Mouse 12A11 Mu12A11 Mu12A11 V_(H) SEQID NO: 31 Mu12A11 V_(L) SEQ ID NO: 41 Humanized 12A11.1b Hu12A11.1bHu12A11.1b V_(H) SEQ ID NO: 32 Hu12A11.1a V_(L) SEQ ID NO: 42 Humanized12A11.2b Hu12A11.2b1 Hu12A11.2b V_(H) SEQ ID NO: 33 Hu12A11.1a V_(L) SEQID NO: 42 with mouse CDRs Humanized 12A11.2b Hu12A11.2b2 Hu12A11.2bV_(H) SEQ ID NO: 34 Hu12A11.1a V_(L) SEQ ID NO: 42 with M99I in CDR-H3M99I variant Humanized 12A11.2b Hu12A11.2b3 Hu12A11.2b V_(H) SEQ ID NO:35 Hu12A11.1a V_(L) SEQ ID NO: 42 with M99V in CDR-H3 M99V variantHumanized 12A11.2b Hu12A11.2b4 Hu12A11.2b V_(H) SEQ ID NO: 36 Hu12A11.1aV_(L) SEQ ID NO: 42 with M99L in CDR-H3 M99L variant

7.2. Definitions

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art.

7.3. Anti-PD-1 Antibodies and Binding Fragments

In one aspect, the disclosure concerns antibodies and/or bindingfragments thereof that specifically bind programmed cell death protein 1(PD-1) receptor (also known as PDCD1, CD279, PD1, SLEB2, or SLE1).

As used herein, the term “antibody” (Ab) refers to an immunoglobulinmolecule that specifically binds to a particular antigen—here, PD-1. Insome embodiments, the anti-PD-1 antibodies of the disclosure bind tohuman PD-1 and thereby modulate the immune system. The resulting immunesystem response is cytotoxic to tumor cells. Anti-PD-1 antibodies of thedisclosure comprise complementarity determining regions (CDRs), alsoknown as hypervariable regions, in both the light chain and the heavychain variable domains. The more highly conserved portions of variabledomains are called the framework (FR). As is known in the art, the aminoacid position/boundary delineating a hypervariable region of an antibodycan vary, depending on the context and the various definitions known inthe art. Some positions within a variable domain may be viewed as hybridhypervariable positions in that these positions can be deemed to bewithin a hypervariable region under one set of criteria while beingdeemed to be outside a hypervariable region under a different set ofcriteria. One or more of these positions can also be found in extendedhypervariable regions. The disclosure provides antibodies comprisingmodifications in these hybrid hypervariable positions. The variabledomains of native heavy and light chains each comprise four FR regions,largely by adopting a β-sheet configuration, connected by three CDRs,which form loops connecting, and in some cases forming part of, theβ-sheet structure. The CDRs in each chain are held together in closeproximity by the FR regions and, with the CDRs from the other chain,contribute to the formation of the target binding site of antibodies.See Kabat et al., Sequences of Proteins of Immunological Interest(National Institute of Health, Bethesda, Md. 1987). As used herein,numbering of immunoglobulin amino acid residues is done according to theimmunoglobulin amino acid residue numbering system of Kabat et al.unless otherwise indicated.

The antibodies of the disclosure may be polyclonal, monoclonal,genetically engineered, and/or otherwise modified in nature, includingbut not limited to chimeric antibodies, humanized antibodies, humanantibodies, single chain antibodies, etc. In various embodiments, theantibodies comprise all or a portion of a constant region of anantibody. In some embodiments, the constant region is an isotypeselected from: IgA (e.g., IgA₁ or IgA₂), IgD, IgE, IgG (e.g., IgG₁,IgG₂, IgG₃ or IgG₄), and IgM. In specific embodiments, the antibodiesdescribed herein comprise an IgG₁. In other embodiments, the anti-PD-1antibodies comprise an IgG₂. In yet other embodiments, the anti-PD-1antibodies comprise an IgG₄. As used herein, the “constant region” of anantibody includes the natural constant region, allotypes or naturalvariants, such as D356E and L358M, or A431G in human IgG₁. See, e.g.,Jefferis and Lefranc, MAbs, 1(4): 332-338 (July-August 2009).

The light constant region of an anti-PD-1 antibody may be a kappa (κ)light region or a lambda (λ) region. A light region can be any one ofthe known subtypes, e.g., λ₁, λ₂, λ₃, or λ₄. In some embodiments, theanti-PD-1 antibody comprises a kappa (κ) light region.

The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. A monoclonal antibodyis derived from a single clone, including any eukaryotic, prokaryotic,or phage clone, by any means available or known in the art. Monoclonalantibodies useful with the present disclosure can be prepared using awide variety of techniques known in the art including the use ofhybridoma, recombinant, and phage display technologies, or a combinationthereof. In many uses of the present disclosure, including in vivo useof the anti-PD-1 antibodies in humans, chimeric, humanized, or humanantibodies can suitably be used.

The term “chimeric” antibody as used herein refers to an antibody havingvariable sequences derived from a non-human immunoglobulin, such as arat or a mouse antibody, and human immunoglobulin constant regions,typically chosen from a human immunoglobulin template. Methods forproducing chimeric antibodies are known in the art. See, e.g., Morrison,1985, Science 229(4719):1202-7; Oi et al., 1986, BioTechniques4:214-221; Gillies et al., 1985, J. Immunol. Methods 125:191-202; U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816,397.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins that contain minimal sequences derived from non-humanimmunoglobulin. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin sequence. The humanizedantibody can also comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin consensussequence. Methods of antibody humanization are known in the art. See,e.g., Riechmann et al., 1988, Nature 332:323-7; U.S. Pat. Nos.5,530,101; 5,585,089; 5,693,761; 5,693,762; and U.S. Pat. No. 6,180,370to Queen et al.; EP239400; PCT publication WO 91/09967; U.S. Pat. No.5,225,539; EP592106; EP519596; Padlan, 1991, Mol. Immunol., 28:489-498;Studnicka et al., 1994, Prot. Eng. 7:805-814; Roguska et al., 1994,Proc. Natl. Acad. Sci. 91:969-973; and U.S. Pat. No. 5,565,332.

“Human antibodies” include antibodies having the amino acid sequence ofa human immunoglobulin and include antibodies isolated from humanimmunoglobulin libraries or from animals transgenic for one or morehuman immunoglobulins and that do not express endogenousimmunoglobulins. Human antibodies can be made by a variety of methodsknown in the art including phage display methods using antibodylibraries derived from human immunoglobulin sequences. See U.S. Pat.Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645; WO98/50433; WO 98/24893; WO 98/16654; WO 96/34096; WO 96/33735; and WO91/10741. Human antibodies can also be produced using transgenic micewhich are incapable of expressing functional endogenous immunoglobulinsbut which can express human immunoglobulin genes. See, e.g., PCTpublications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; U.S.Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016;5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598. In addition,companies such as LakePharma, Inc. (Belmont, Calif.) or Creative BioLabs(Shirley, N.Y.) can be engaged to provide human antibodies directedagainst a selected antigen using technology similar to that describedabove. Fully human antibodies that recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach, a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope (see, Jespers et al., 1988, Biotechnology12:899-903).

Anti-PD-1 antibodies of the disclosure include full-length (intact)antibody molecules.

In some embodiments, the present disclosure also includes anti-PD-1binding fragments that are capable of specifically binding PD-1.Examples of antibody binding fragments include by way of example and notlimitation, Fab, Fab′, F(ab′)₂, Fv fragments, single chain Fv fragmentsand single domain fragments.

An Fab fragment contains the constant domain of the light chain and thefirst constant domain (CH1) of the heavy chain. Fab′ fragments differfrom Fab fragments by the addition of a few residues at the carboxylterminus of the heavy chain CH1 domain including one or more cysteinesfrom the antibody hinge region. F(ab′) fragments are produced bycleavage of the disulfide bond at the hinge cysteines of the F(ab′)₂pepsin digestion product. Additional chemical couplings of antibodyfragments are known to those of ordinary skill in the art. Fab andF(ab′)₂ fragments lack the Fc fragment of an intact antibody, clear morerapidly from the circulation of animals, and may have less non-specifictissue binding than an intact antibody (see, e.g., Wahl et al., 1983, J.Nucl. Med. 24:316).

An “Fv” fragment is the minimum fragment of an antibody that contains acomplete target recognition and binding site. This region consists of adimer of one heavy and one light chain variable domain in a tight,non-covalent association (V_(H)-V_(L) dimer). It is in thisconfiguration that the three CDRs of each variable domain interact todefine a target binding site on the surface of the V_(H)-V_(L) dimer.Often, the six CDRs confer target binding specificity to the antibody.However, in some instances even a single variable domain (or half of anFv comprising only three CDRs specific for a target) can have theability to recognize and bind target, although at a lower affinity thanthe entire binding site.

“Single-chain Fv” or “scFv” antibody binding fragments comprise theV_(H) and V_(L) domains of an antibody, where these domains are presentin a single polypeptide chain. Generally, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the scFv to form the desired structure for target binding.

“Single domain antibodies” are composed of a single V_(H) or V_(L)domains which exhibit sufficient affinity to PD-1. In a specificembodiment, the single domain antibody is a camelized antibody (See,e.g., Riechmann, 1999, Journal of Immunological Methods 231:25-38).

The anti-PD-1 antibodies of the disclosure include derivatizedantibodies. For example, but not by way of limitation, derivatizedantibodies are typically modified by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein. Any of numerous chemical modifications can becarried out by known techniques, including, but not limited to, specificchemical cleavage, acetylation, formylation, metabolic synthesis oftunicamycin, etc. Additionally, the derivative can contain one or morenon-natural amino acids, e.g., using ambrx technology (See, e.g.,Wolfson, 2006, Chem. Biol. 13(10):1011-2).

The anti-PD-1 antibodies or binding fragments may be antibodies orfragments whose sequences have been modified to alter at least oneconstant region-mediated biological effector function. For example, insome embodiments, an anti-PD-1 antibody may be modified to reduce atleast one constant region-mediated biological effector function relativeto the unmodified antibody, e.g., reduced binding to one or more of theFc receptors (FcγR) such as FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA and/orFcγRIIIB. FcγR binding can be reduced by mutating the immunoglobulinconstant region segment of the antibody at particular regions necessaryfor FcγR interactions (See, e.g., Canfield and Morrison, 1991, J. Exp.Med. 173:1483-1491; and Lund et al., 1991, J. Immunol. 147:2657-2662).Reduction in FcγR binding ability of the antibody can also reduce othereffector functions which rely on FcγR interactions, such asopsonization, phagocytosis and antigen-dependent cellular cytotoxicity(“ADCC”).

The anti-PD-1 antibody or binding fragment described herein includeantibodies that have been modified to acquire or improve at least oneconstant region-mediated biological effector function relative to anunmodified antibody, e.g., to enhance FcγR interactions (See, e.g., USPatent Appl. No. 2006/0134709). For example, an anti-PD-1 antibody ofthe disclosure can have a constant region that binds FcγRI, FcγRIIA,FcγRIIB, FcγRIIIA and/or FcγRIIIB with greater affinity than thecorresponding wild type constant region.

Thus, antibodies of the disclosure may have alterations in biologicalactivity that result in increased or decreased opsonization,phagocytosis, or ADCC. Such alterations are known in the art. Forexample, modifications in antibodies that reduce ADCC activity aredescribed in U.S. Pat. No. 5,834,597. An exemplary ADCC lowering variantcorresponds to “mutant 3” (also known as “M3,” shown in FIG. 4 of U.S.Pat. No. 5,834,597) in which residues 234 and 237 (using EU numbering)are substituted with alanines. A mutant 3 (also known as “M3”) variationmay be used in a number of antibody isotypes, e.g., IgG₂.

Additional substitutions that can modify FcγR binding and/or ADCCeffector function of an anti-PD-1 antibody include the K322Asubstitution or the L234A and L235A double substitution in the Fcregion. See, e.g., Hezareh, et al. J. Virol., 75 (24): 12161-12168(2001).

In some embodiments, the anti-PD-1 antibodies of the disclosure have lowlevels of, or lack, fucose. Antibodies lacking fucose have beencorrelated with enhanced ADCC activity, especially at low doses ofantibody. See Shields et al., 2002, J. Biol. Chem. 277:26733-26740;Shinkawa et al., 2003, J. Biol. Chem. 278:3466-73. Methods of preparingfucose-less antibodies include growth in rat myeloma YB2/0 cells (ATCCCRL 1662). YB2/0 cells express low levels of FUT8 mRNA, which encodesα-1,6-fucosyltransferase, an enzyme necessary for fucosylation ofpolypeptides.

The anti-PD-1 antibodies of the disclosure can comprise modified (orvariant) CH2 domains or entire Fc domains that include amino acidsubstitutions that increase binding to FcγRIIB and/or reduced binding toFcγRIIIA as compared to the binding of a corresponding wild-type CH2 orFc region. Variant CH2 or variant Fc domains have been described in U.S.Patent Appl. No. 2014/0377253. A variant CH2 or variant Fc domaintypically includes one or more substitutions at position 263, position266, position 273, and position 305, wherein the numbering of theresidues in the Fc domain is that of the EU index as in Kabat. In someembodiments, the anti-PD-1 antibodies comprise one or more substitutionsselected from V263L, V266L, V273C, V273E, V273F, V273L, V273M, V273S,V273Y, V305K, and V305W, relative to the wild-type CH2 domain. Inspecific embodiments, the one or more substitutions of the CH2 domainare selected from V263L, V273E, V273F, V273M, V273S, and V273Y, relativeto the CH2 domain of a human IgG₁. For example, the one or moresubstitutions of an IgG₁ CH2 domain can be V273E. In another specificembodiment, the anti-PD-1 antibody of the disclosure comprises a variantIgG₁ hinge region comprising the amino acid substitution V263L.

Other examples of variant CH2 or variant Fc domains that can affordincreased binding to FcγRIIB and/or reduced binding to FcγRIIIA ascompared to the binding of a corresponding wild-type CH2 or Fc regioninclude those found in Vonderheide, et al. Clin. Cancer Res., 19(5),1035-1043 (2013), such as S267E or S267E/L328F in human IgG₁.

Anti-PD-1 antibodies or binding fragments that comprise a human IgG₄constant region can comprise the S228P mutation, which has been reportedto prevent Fab arm exchange. See, e.g., Silva, J P et al. Journal ofBiological Chemistry, 290(9), 5462-5469 (2015).

In some embodiments, the anti-PD-1 antibodies or binding fragmentsinclude modifications that increase or decrease their binding affinitiesto the fetal Fc receptor, FcRn, for example, by mutating theimmunoglobulin constant region segment at particular regions involved inFcRn interactions (see, e.g., WO 2005/123780). In particularembodiments, an anti-PD-1 antibody of the IgG class is mutated such thatat least one of amino acid residues 250, 314, and 428 of the heavy chainconstant region is substituted alone, or in any combinations thereof,such as at positions 250 and 428, or at positions 250 and 314, or atpositions 314 and 428, or at positions 250, 314, and 428, with positions250 and 428 a specific combination. For position 250, the substitutingamino acid residue can be any amino acid residue other than threonine,including, but not limited to, alanine, cysteine, aspartic acid,glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine,leucine, methionine, asparagine, proline, glutamine, arginine, serine,valine, tryptophan, or tyrosine. For position 314, the substitutingamino acid residue can be any amino acid residue other than leucine,including, but not limited to, alanine, cysteine, aspartic acid,glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine,methionine, asparagine, proline, glutamine, arginine, serine, threonine,valine, tryptophan, or tyrosine. For position 428, the substitutingamino acid residues can be any amino acid residue other than methionine,including, but not limited to, alanine, cysteine, aspartic acid,glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine,leucine, asparagine, proline, glutamine, arginine, serine, threonine,valine, tryptophan, or tyrosine. An exemplary substitution known tomodify Fc effector function is the Fc substitution M428L, which canoccur in combination with the Fc substitution T250Q. Additional specificcombinations of suitable amino acid substitutions are identified inTable 1 of U.S. Pat. No. 7,217,797. Such mutations increase binding toFcRn, which protects the antibody from degradation and increases itshalf-life.

An anti-PD-1 antibody may have one or more amino acids inserted into oneor more of its CDRs, for example as described in Jung and Plückthun,1997, Protein Engineering 10:9, 959-966; Yazaki et al., 2004, ProteinEng. Des Sel. 17(5):481-9. Epub 2004 Aug. 17; and U.S. Pat. Appl. No.2007/0280931.

Anti-PD-1 antibodies with high affinity for human PD-1 may be desirablefor therapeutic and diagnostic uses. Accordingly, the present disclosurecontemplates antibodies having a high binding affinity to human PD-1. Inspecific embodiments, the anti-PD-1 antibodies that bind human PD-1 withan affinity of at least about 100 nM, but may exhibit higher affinity,for example, at least about 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30nM, 25 nM, 20 nM, 15 nM, 10 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1nM, 0.1 nM, 0.01 nM, or even higher. In some embodiments, the antibodiesbind human PD-1 with an affinity in the range of about 1 pM to about 100nM, or an affinity ranging between any of the foregoing values.

Affinity of anti-PD-1 antibodies for human PD-1 can be determined usingtechniques well known in the art or described herein, such as forexample, but not by way of limitation, ELISA, isothermal titrationcalorimetry (ITC), surface plasmon resonance, or fluorescentpolarization assay.

Anti-PD-1 antibodies generally comprise a heavy chain comprising avariable region (V_(H)) having three complementarity determining regions(“CDRs”) referred to herein (in N→C order) as V_(H) CDR #1, V_(H) CDR#2, and V_(H) CDR #3, and a light chain comprising a variable region(V_(L)) having three complementarity determining regions referred toherein (in N→C order) as V_(L) CDR #1, V_(L) CDR #2, and V_(L) CDR #3.The amino acid sequences of exemplary CDRs, as well as the amino acidsequence of the V_(H) and V_(L) regions of the heavy and light chains ofexemplary anti-PD-1 are provided herein. Specific embodiments ofanti-PD-1 antibodies include these exemplary CDRs and/or V_(H) and/orV_(L) sequences, as well as antibodies that compete for binding humanPD-1 with such antibodies.

In some embodiments, an anti-PD-1 antibody is suitable foradministration to humans. In a specific embodiment, the anti-PD-1antibody is humanized. In some embodiments, the amino acid sequences ofthe CDRs of an anti-PD-1 antibody are selected from the followingsequences:

CDR Sequence (N→C) Identifier V_(H) CDR#1: GYTFTHYGMN (SEQ ID NO: 11)V_(H) CDR#2: WVNTYTGEPTYADDFKG (SEQ ID NO: 12) V_(H) CDR#3: EGEGLGFGD(SEQ ID NO: 13) EGEGIGFGD (SEQ ID NO: 21) EGEGVGFGD (SEQ ID NO: 22)EGEGMGFGD (SEQ ID NO: 23) V_(L) CDR#1: RSSQSIVHSHGDTYLE (SEQ ID NO: 14)V_(L) CDR#2: KVSNRFS (SEQ ID NO: 15) V_(L) CDR#3: FQGSHIPVT(SEQ ID NO: 16)

Specific exemplary embodiments of anti-PD-1 antibodies with the aboveCDRs are described herein. In some embodiments, an anti-PD-1 antibodyhas the CDRs of SEQ ID NOS: 11, 12, 13, 14, 15, and 16. In someembodiments, an anti-PD-1 antibody has the CDRs of SEQ ID NOS: 11, 12,21, 14, 15, and 16. In some embodiments, an anti-PD-1 antibody has theCDRs of SEQ ID NOS: 11, 12, 22, 14, 15, and 16. In some embodiments, ananti-PD-1 antibody has the CDRs of SEQ ID NOS: 11, 12, 23, 14, 15, and16.

In some embodiments, each CDR of an anti-PD-1 antibody, independently ofthe others, is selected to correspond in sequence to the respective CDRof an antibody provided in TABLE 3. In some embodiments, an anti-PD-1antibody is an IgG, and has a V_(H) and V_(L) corresponding in sequenceto the V_(H) and V_(L) of an antibody provided in TABLE 3.

In some embodiments, an anti-PD-1 antibody comprises a V_(H) chaincorresponding in sequence to SEQ ID NO:31; and a V_(L) chaincorresponding in sequence to SEQ ID NO:41. In some embodiments, ananti-PD-1 antibody comprises a V_(H) chain corresponding in sequence toSEQ ID NO:32; and a V_(L) chain corresponding in sequence to SEQ IDNO:42. In some embodiments, an anti-PD-1 antibody comprises a V_(H)chain corresponding in sequence to SEQ ID NO:33; and a V_(L) chaincorresponding in sequence to SEQ ID NO:42. In some embodiments, ananti-PD-1 antibody comprises a V_(H) chain corresponding in sequence toSEQ ID NO:34; and a V_(L) chain corresponding in sequence to SEQ IDNO:42. In some embodiments, an anti-PD-1 antibody comprises a V_(H)chain corresponding in sequence to SEQ ID NO:35; and a V_(L) chaincorresponding in sequence to SEQ ID NO:42. In some embodiments, ananti-PD-1 antibody comprises a V_(H) chain corresponding in sequence toSEQ ID NO:36; and a V_(L) chain corresponding in sequence to SEQ IDNO:42.

Certain mutations of a V_(H) or V_(L) sequence in an anti-PD-1 antibodydescribed herein would be understood by a person of skill to affordanti-PD-1 antibodies within the scope of the disclosure. Mutations mayinclude amino acid substitutions, additions, or deletions from a V_(H)or V_(L) sequence as disclosed herein while retaining significantanti-PD-1 activity. Accordingly, in some embodiments, an anti-PD-1antibody comprises a V_(H) sequence having at least 85%, at least 90%,at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% sequence identity to the V_(H) sequence of any one of theantibodies shown in TABLE 3. An anti-PD-1 antibody can comprise a V_(H)sequence having up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, orup to 2 mutations compared with the V_(H) sequence of any one of theantibodies shown in TABLE 3. In some embodiments, an anti-PD-1 antibodycan comprise a V_(H) sequence having 5 or fewer, 4 or fewer, 3 or fewer,or 2 or fewer mutations compared with the V_(H) sequence of any one ofthe antibodies shown in TABLE 3. In some embodiments, an anti-PD-1antibody comprises a V_(L) sequence having at least 85%, at least 90%,at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% sequence identity to the V_(L) sequence of any one of theantibodies shown in TABLE 3. An anti-PD-1 antibody can comprise a V_(L)sequence having up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, orup to 2 mutations compared with the V_(L) sequence of any one of theantibodies shown in TABLE 3. In some embodiments, an anti-PD-1 antibodycan comprise a V_(L) sequence having 5 or fewer, 4 or fewer, 3 or fewer,or 2 or fewer mutations compared with the V_(L) sequence of any one ofthe antibodies shown in TABLE 3.

In some embodiments, an anti-PD-1 antibody comprises a heavy chain aminoacid sequence according to:

(SEQ ID NO: 51) EIQLVQSGAEVKKPGSSVKVSCKASGYTFTHYGMNWVRQAPGQGLEWVGWVNTYTGEPTYADDFKGRLTFTLDTSTSTAYMELSSLRSEDTAVYYCTREGEGLGFGDWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;and a light chain amino acid sequence according to:

(SEQ ID NO: 61) DVVMTQSPLSLPVTPGEPASISCRSSQSIVHSHGDTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPVTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC,wherein the underlined amino acids represent the CDRs and the italicizedamino acids represent the constant regions.

Post-translational modifications to the sequences of an anti-PD-1antibody may occur, such as cleavage of one or more (e.g., 1, 2, 3, ormore) amino acid residues on the C-terminal end of the antibody heavychain.

In some embodiments, an anti-PD-1 antibody comprises a heavy chain aminoacid sequence according to:

(SEQ ID NO: 52) EIQLVQSGAEVKKPGSSVKVSCKASGYTFTHYGMNWVRQAPGQGLEWVGWVNTYTGEPTYADDFKGRLTFTLDTSTSTAYMELSSLRSEDTAVYYCTREGEGLGFGDWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG;and a light chain amino acid sequence according to:

(SEQ ID NO: 61) DVVMTQSPLSLPVTPGEPASISCRSSQSIVHSHGDTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPVTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC,wherein the underlined amino acids represent the CDRs and the italicizedamino acids represent the constant regions.

Additional post-translational modifications of an anti-PD-1 antibody mayinclude glycosylation. Common biantennary complexes can be composed of acore structure having two N-acetylglucosamine (GlcNAc), three mannose,and two GlcNAc residues that are β-1,2 linked to α-6 mannose and α-3mannose to form two antennae. One or more fucose (Fuc), galactose (Gal),high mannose glycans Man-5 or Man-9, bisecting GlcNAc, and sialic acidincluding N-acetylneuraminic acid (NANA) or N-glycolylneuraminic acid(NGNA) residues may be attached to the core. N-linked glycoforms mayinclude G0 (protein having a core biantennary glycosylation structure),G0F (fucosylated G0), G0F GlcNAc, G1 (protein having a coreglycosylation structure with one galactose residue), G1F (fucosylatedG1), G2 (protein having a core glycosylation structure with twogalactose residues), and/or G2F (fucosylated G2). In some embodiments,an anti-PD-1 antibody has a G0F glycan.

In some embodiments, the anti-PD-1 antibodies compete for binding humanPD-1 in in vitro assays with a reference antibody. In some embodiments,the anti-PD-1 antibodies compete for binding human PD-1 on cellsexpressing human PD-1. The reference antibody may be any of theanti-PD-1 antibodies described herein. In some embodiments, thereference antibody is an antibody provided in TABLE 3. In specificembodiments, the reference antibody is selected from mouse antibody12A11 (“Mu12A11”). In some embodiments, the reference antibody is ahumanized version of Mu12A11. In a specific embodiment, the referenceantibody is humanized antibody 12A11.1b (“Hu12A11.1b”) or humanizedantibody 12A11.2b M99L (“Hu12A11.2b4”).

In some embodiments, the anti-PD-1 antibodies antagonize, e.g., inhibit,human PD-1 (SEQ ID NO:1). PD-1 receptor antagonism can occur by a numberof mechanisms, for example, by inhibiting binding of PD-1 by human PD-L1(SEQ ID NO:3) or PD-L2 (SEQ ID NO:4).

The anti-PD-1 antibodies described herein generally bind specifically tohuman PD-1. Cross reactivity of the antibodies for binding to PD-1 fromother species, for example, from monkey, e.g., cynomolgus monkey, mayoffer advantages, such as the ability to test in monkey animal modelsfor biological activity. Such animal model testing may be used to screenanti-PD-1 antibodies to select properties related to efficacy, e.g.,favorable pharmacokinetics, or those related to safety, e.g., decreasedhepatic toxicity. In some embodiments, the anti-PD-1 antibodies bind tocynomolgus PD-1 as well as human PD-1.

Assays for competition include, but are not limited to, a radioactivematerial labeled immunoassay (RIA), an enzyme-linked immunosorbent assay(ELISA), a sandwich ELISA, fluorescence activated cell sorting (FACS)assays, and surface plasmon resonance assays.

In conducting an antibody competition assay between a reference antibodyand a test antibody (irrespective of species or isotype), one may firstlabel the reference with a detectable label, such as a fluorophore,biotin or an enzymatic (or even radioactive) label to enable subsequentidentification. In this case, cells expressing human PD-1 are incubatedwith unlabeled test antibody, labeled reference antibody is added, andthe intensity of the bound label is measured. If the test antibodycompetes with the labeled reference antibody by binding to anoverlapping epitope, the intensity will be decreased relative to acontrol reaction carried out without test antibody.

In a specific embodiment of this assay, the concentration of labeledreference antibody that yields 80% of maximal binding (“conc_(80%)”)under the assay conditions (e.g., a specified density of cells) is firstdetermined, and a competition assay carried out with 10× conc_(80%) ofunlabeled test antibody and conc_(80%) of labeled reference antibody.

The inhibition can be expressed as an inhibition constant, or K which iscalculated according to the following formula:

K _(i) =IC ₅₀/(1+[reference Ab concentration]/K _(d)),

where IC₅₀ is the concentration of test antibody that yields a 50%reduction in binding of the reference antibody and K_(d) is thedissociation constant of the reference antibody, a measure of itsaffinity for human PD-1. Antibodies that compete with anti-PD-1antibodies disclosed herein can have a K_(i) from 10 pM to 10 nM underassay conditions described herein.

In various embodiments, a test antibody is considered to compete with areference antibody if it decreases binding of the reference antibody byat least about 20% or more, for example, by at least about 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95% or even more, or by a percentageranging between any of the foregoing values, at a reference antibodyconcentration that is 80% of maximal binding under the specific assayconditions used, and a test antibody concentration that is 10-foldhigher than the reference antibody concentration.

A specific assay and assay conditions useful for assessing whether anantibody competes for binding human PD-1 with a reference antibody asdescribed herein is provided in Section 8.1.5.

7.4. Polynucleotides Encoding the Anti-PD-1 Antibodies, ExpressionSystems and Methods of Making the Antibodies

The present disclosure encompasses nucleic acid molecules encodingimmunoglobulin light and heavy chain genes for anti-PD-1 antibodies,vectors comprising such nucleic acids, and host cells capable ofproducing the anti-PD-1 antibodies of the disclosure.

An anti-PD-1 antibody of the disclosure can be prepared by recombinantexpression of immunoglobulin light and heavy chain genes in a host cell.To express an antibody recombinantly, a host cell is transfected withone or more recombinant expression vectors carrying DNA fragmentsencoding the immunoglobulin light and heavy chains of the antibody suchthat the light and heavy chains are expressed in the host cell and,optionally, secreted into the medium in which the host cells arecultured, from which medium the antibodies can be recovered. Standardrecombinant DNA methodologies are used to obtain antibody heavy andlight chain genes, incorporate these genes into recombinant expressionvectors and introduce the vectors into host cells, such as thosedescribed in Molecular Cloning; A Laboratory Manual, Second Edition(Sambrook, Fritsch and Maniatis (eds), Cold Spring Harbor, N.Y., 1989),Current Protocols in Molecular Biology (Ausubel, F. M. et al., eds.,Greene Publishing Associates, 1989) and in U.S. Pat. No. 4,816,397.

To generate nucleic acids encoding such anti-PD-1 antibodies, DNAfragments encoding the light and heavy chain variable regions are firstobtained. These DNAs can be obtained by amplification and modificationof germline DNA or cDNA encoding light and heavy chain variablesequences, for example using the polymerase chain reaction (PCR).Germline DNA sequences for human heavy and light chain variable regiongenes are known in the art (See, e.g., the “VBASE” human germlinesequence database; see also Kabat, E. A. et al., 1991, Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242; Tomlinson etal., 1992, J. Mol. Biol. 22T:116-198; and Cox et al., 1994, Eur. J.Immunol. 24:827-836; the contents of each of which are incorporatedherein by reference).

Once DNA fragments encoding anti-PD-1 antibody-related V_(H) and V_(L)segments are obtained, these DNA fragments can be further manipulated bystandard recombinant DNA techniques, for example to convert the variableregion genes to full-length antibody chain genes, to Fab fragment genesor to a scFv gene. In these manipulations, a V_(L)- or V_(H)-encodingDNA fragment is operatively linked to another DNA fragment encodinganother protein, such as an antibody constant region or a flexiblelinker. The term “operatively linked,” as used in this context, isintended to mean that the two DNA fragments are joined such that theamino acid sequences encoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the V_(H) region can be converted to afull-length heavy chain gene by operatively linking the V_(H)-encodingDNA to another DNA molecule encoding heavy chain constant regions (CH1,CH2, CH3 and, optionally, CH4). The sequences of human heavy chainconstant region genes are known in the art (See, e.g., Kabat, E. A., etal., 1991, Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The heavy chain constant regioncan be an IgG₁, IgG₂, IgG₃, IgG₄, IgA, IgE, IgM or IgD constant region,but in certain embodiments is an IgG₁ or IgG₄. For a Fab fragment heavychain gene, the V_(H)-encoding DNA can be operatively linked to anotherDNA molecule encoding only the heavy chain CH1 constant region.

The isolated DNA encoding the V_(L) region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the V_(L)-encoding DNA to another DNA moleculeencoding the light chain constant region, CL. The sequences of humanlight chain constant region genes are known in the art (See, e.g.,Kabat, et al., 1991, Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242) and DNA fragments encompassing these regionscan be obtained by standard PCR amplification. The light chain constantregion can be a kappa or lambda constant region, but in certainembodiments is a kappa constant region. To create a scFv gene, theV_(H)- and V_(L)-encoding DNA fragments are operatively linked toanother fragment encoding a flexible linker, e.g., encoding the aminoacid sequence (Gly₄˜Ser)₃ (SEQ ID NO:5), such that the V_(H) and V_(L)sequences can be expressed as a contiguous single-chain protein, withthe V_(L) and V_(H) regions joined by the flexible linker (See, e.g.,Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl.Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990, Nature348:552-554).

To express the anti-PD-1 antibodies of the disclosure, DNAs encodingpartial or full-length light and heavy chains, obtained as describedabove, are inserted into expression vectors such that the genes areoperatively linked to transcriptional and translational controlsequences. In this context, the term “operatively linked” is intended tomean that an antibody gene is ligated into a vector such thattranscriptional and translational control sequences within the vectorserve their intended function of regulating the transcription andtranslation of the antibody gene. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. The antibody light chain gene and the antibody heavy chaingene can be inserted into separate vectors or, more typically, bothgenes are inserted into the same expression vector.

The antibody genes are inserted into the expression vector by standardmethods (e.g., ligation of complementary restriction sites on theantibody gene fragment and vector, or blunt end ligation if norestriction sites are present). Prior to insertion of the anti-PD-1antibody-related light or heavy chain sequences, the expression vectorcan already carry antibody constant region sequences. For example, oneapproach to converting the anti-PD-1 monoclonal antibody-related V_(H)and V_(L) sequences to full-length antibody genes is to insert them intoexpression vectors already encoding heavy chain constant and light chainconstant regions, respectively, such that the V_(H) segment isoperatively linked to the CH segment(s) within the vector and the V_(L)segment is operatively linked to the CL segment within the vector.Additionally or alternatively, the recombinant expression vector canencode a signal peptide that facilitates secretion of the antibody chainfrom a host cell. The antibody chain gene can be cloned into the vectorsuch that the signal peptide is linked in-frame to the amino terminus ofthe antibody chain gene. The signal peptide can be an immunoglobulinsignal peptide or a heterologous signal peptide (i.e., a signal peptidefrom a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the disclosure carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif., 1990. It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Suitable regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. For furtherdescription of viral regulatory elements, and sequences thereof, see,e.g., U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 byBell et al., and U.S. Pat. No. 4,968,615 by Schaffner et al.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the disclosure can carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (See, e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Suitable selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in DHFR⁻ host cells withmethotrexate selection/amplification) and the neo gene (for G418selection). For expression of the light and heavy chains, the expressionvector(s) encoding the heavy and light chains is transfected into a hostcell by standard techniques. The various forms of the term“transfection” are intended to encompass a wide variety of techniquescommonly used for the introduction of exogenous DNA into a prokaryoticor eukaryotic host cell, e.g., electroporation, lipofection,calcium-phosphate precipitation, DEAE-dextran transfection and the like.

It is possible to express the antibodies of the disclosure in eitherprokaryotic or eukaryotic host cells. In certain embodiments, expressionof antibodies is performed in eukaryotic cells, e.g., mammalian hostcells, of optimal secretion of a properly folded and immunologicallyactive antibody. Exemplary mammalian host cells for expressing therecombinant antibodies of the disclosure include Chinese Hamster Ovary(CHO cells) (including DHFR⁻ CHO cells, described in Urlaub and Chasin,1980, Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFRselectable marker, e.g., as described in Kaufman and Sharp, 1982, Mol.Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells. Whenrecombinant expression vectors encoding antibody genes are introducedinto mammalian host cells, the antibodies are produced by culturing thehost cells for a period of time sufficient to allow for expression ofthe antibody in the host cells or secretion of the antibody into theculture medium in which the host cells are grown. Antibodies can berecovered from the culture medium using standard protein purificationmethods. Host cells can also be used to produce portions of intactantibodies, such as Fab fragments or scFv molecules. It is understoodthat variations on the above procedure are within the scope of thepresent disclosure. For example, it can be desirable to transfect a hostcell with DNA encoding either the light chain or the heavy chain (butnot both) of an anti-PD-1 antibody of this disclosure.

Recombinant DNA technology can also be used to remove some or all of theDNA encoding either or both of the light and heavy chains that is notnecessary for binding to human PD-1. The molecules expressed from suchtruncated DNA molecules are also encompassed by the antibodies of thedisclosure.

For recombinant expression of an anti-PD-1 antibody of the disclosure,the host cell can be co-transfected with two expression vectors of thedisclosure, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors can contain identical selectable markers, or they can eachcontain a separate selectable marker. Alternatively, a single vector canbe used which encodes both heavy and light chain polypeptides.

Once a nucleic acid encoding one or more portions of an anti-PD-1antibody has been obtained, further alterations or mutations can beintroduced into the coding sequence, for example to generate nucleicacids encoding antibodies with different CDR sequences, antibodies withreduced affinity to the Fc receptor, or antibodies of differentsubclasses.

The anti-PD-1 antibodies of the disclosure can also be produced bychemical synthesis (e.g., by the methods described in Solid PhasePeptide Synthesis, 2^(nd) ed., 1984 The Pierce Chemical Co., Rockford,Ill.). Variant antibodies can also be generated using a cell-freeplatform (See, e.g., Chu et al., Biochemia No. 2, 2001 (Roche MolecularBiologicals) and Murray et al., 2013, Current Opinion in ChemicalBiology, 17:420-426).

Once an anti-PD-1 antibody of the disclosure has been produced byrecombinant expression, it can be purified by any method known in theart for purification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Further, theanti-PD-1 antibodies of the present disclosure can be fused toheterologous polypeptide sequences described herein or otherwise knownin the art to facilitate purification.

Once isolated, the anti-PD-1 antibody can, if desired, be furtherpurified, e.g., by high performance liquid chromatography (see, e.g.,Fisher, Laboratory Techniques In Biochemistry And Molecular Biology,Work and Burdon, eds., Elsevier, 1980), or by gel filtrationchromatography on a Superdex™ 75 column (Pharmacia Biotech AB, Uppsala,Sweden).

7.5. Pharmaceutical Compositions

The anti-PD-1 antibodies described herein may be in the form ofcompositions comprising the antibody and one or more carriers,excipients and/or diluents. The compositions may be formulated forspecific uses, such as for veterinary uses or pharmaceutical uses inhumans. The form of the composition (e.g., dry powder, liquidformulation, etc.) and the excipients, diluents and/or carriers usedwill depend upon the intended uses of the antibody and, for therapeuticuses, the mode of administration.

For therapeutic uses, the compositions may be supplied as part of asterile, pharmaceutical composition that includes a pharmaceuticallyacceptable carrier. This composition can be in any suitable form(depending upon the desired method of administering it to a patient).The pharmaceutical composition can be administered to a patient by avariety of routes such as orally, transdermally, subcutaneously,intranasally, intravenously, intramuscularly, intratumorally,intrathecally, topically or locally. The most suitable route foradministration in any given case will depend on the particular antibody,the subject, and the nature and severity of the disease and the physicalcondition of the subject. Typically, the pharmaceutical composition willbe administered intravenously or subcutaneously.

Pharmaceutical compositions can be conveniently presented in unit dosageforms containing a predetermined amount of an anti-PD-1 antibodydescribed herein per dose. The quantity of anti-PD-1 antibody includedin a unit dose will depend on the disease being treated, as well asother factors as are well known in the art. Such unit dosages may be inthe form of a lyophilized dry powder containing an amount of antibodysuitable for a single administration, or in the form of a liquid. Drypowder unit dosage forms may be packaged in a kit with a syringe, asuitable quantity of diluent and/or other components useful foradministration. Unit dosages in liquid form may be conveniently suppliedin the form of a syringe pre-filled with a quantity of the anti-PD-1antibody suitable for a single administration.

The pharmaceutical compositions may also be supplied in bulk formcontaining quantities of anti-PD-1 antibody suitable for multipleadministrations.

Pharmaceutical compositions may be prepared for storage as lyophilizedformulations or aqueous solutions by mixing an antibody having thedesired degree of purity with optional pharmaceutically-acceptablecarriers, excipients or stabilizers typically employed in the art (allof which are referred to herein as “carriers”), i.e., buffering agents,stabilizing agents, preservatives, isotonifiers, non-ionic detergents,antioxidants, and other miscellaneous additives. See, Remington'sPharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additivesshould be nontoxic to the recipients at the dosages and concentrationsemployed.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They may be present at a wide variety ofconcentrations, but will typically be present in concentrations rangingfrom about 2 mM to about 50 mM. Suitable buffering agents for use withthe present disclosure include both organic and inorganic acids andsalts thereof such as citrate buffers (e.g., monosodium citrate-disodiumcitrate mixture, citric acid-trisodium citrate mixture, citricacid-monosodium citrate mixture, etc.), succinate buffers (e.g.,succinic acid-monosodium succinate mixture, succinic acid-sodiumhydroxide mixture, succinic acid-disodium succinate mixture, etc.),tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaricacid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture,etc.), phosphate buffers (e.g., phosphoric acid-monosodium phosphatemixture, phosphoric acid-disodium phosphate mixture, monosodiumphosphate-disodium phosphate mixture, etc.), gluconate buffers (e.g.,gluconic acid-sodium gluconate mixture, gluconic acid-sodium hydroxidemixture, gluconic acid-potassium gluconate mixture, etc.), oxalatebuffer (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodiumhydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactatebuffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodiumhydroxide mixture, lactic acid-potassium lactate mixture, etc.) andacetate buffers (e.g., acetic acid-sodium acetate mixture, aceticacid-sodium hydroxide mixture, etc.). Additionally, fumarate buffers,histidine buffers and trimethylamine salts such as2-amino-2-hydroxymethyl-propane-1,3-diol (i.e., Tris, THAM, ortris(hydroxymethyl)aminomethane) can be used.

Isotonicifiers sometimes known as “stabilizers” can be added to ensureisotonicity of liquid compositions of the present disclosure and includepolyhydric sugar alcohols, for example trihydric or higher sugaralcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol andmannitol. Stabilizers refer to a broad category of excipients which canrange in function from a bulking agent to an additive which solubilizesthe therapeutic agent or helps to prevent denaturation or adherence tothe container wall. Typical stabilizers can be polyhydric sugar alcohols(enumerated above); amino acids such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, ornithine, leucine,2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinositol, galactitol, glycerol and the like,including cyclitols such as inositol; polyethylene glycol; amino acidpolymers; sulfur containing reducing agents, such as urea, glutathione,thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglyceroland sodium thiosulfate; low molecular weight polypeptides (e.g.,peptides of 10 residues or fewer); hydrophilic polymers, such aspolyvinylpyrrolidone monosaccharides, such as xylose, mannose, fructose,glucose; disaccharides such as lactose, maltose, sucrose and trehalose;and trisaccacharides such as raffinose; and polysaccharides such asdextran. Stabilizers may be present in amounts ranging from 0.5 to 10weight % per weight of anti-PD-1 antibody.

Non-ionic surfactants or detergents (also known as “wetting agents”) maybe added to help solubilize the glycoprotein as well as to protect theglycoprotein against agitation-induced aggregation, which also permitsthe formulation to be exposed to shear surface stressed without causingdenaturation of the protein. Suitable non-ionic surfactants includepolysorbates (20, 80, etc.), poloxamers (184, 188 etc.), and pluronicpolyols. Non-ionic surfactants may be present in a range of about 0.05mg/mL to about 1.0 mg/mL.

A specific exemplary embodiment of an aqueous composition suitable foradministration via intravenous infusion comprises 20 mg/mL of ananti-PD-1 antibody comprising a heavy chain sequence of SEQ ID NO:51 orSEQ ID NO:52, and a light chain sequence of SEQ ID NO:61, 15 mMhistidine pH 5.7, 8.0% (w/v) sucrose, and 0.05% (w/v) polysorbate 80.The composition may be in the form of a lyophilized powder that, uponreconstitution with sterile water or other solution suitable forinjection or infusion (for example, 0.9% saline, Ringer's solution,lactated Ringer's solution, etc.) provides the above aqueouscomposition. The composition, or other embodiments of compositions, mayalso be in the form of a syringe or other device suitable for injectionand/or infusion pre-filled with a quantity of composition suitable for asingle administration of the anti-PD-1 antibody.

7.6. Methods of Use

7.6.1. Therapeutic Benefit

Data provided herein demonstrate that the disclosed anti-PD-1 antibodiesantagonize PD-1 in the presence of cancer cells and exert potentanticancer activity against cancers such as solid tumors and bloodmalignancies in vivo. Accordingly, the anti-PD-1 antibodies, bindingfragments, and/or pharmaceutical compositions comprising the anti-PD-1antibodies may be used therapeutically to treat solid tumors or bloodmalignancies.

In some embodiments, the method involves administering to a humanpatient having a solid tumor an amount of an anti-PD-1 antibody thatantagonizes PD-1, and kills tumor cells at a rate effective to providetherapeutic benefit. Solid tumors that may be treated with the anti-PD-1antibody include, but are not limited to, adrenal cancers, bladdercancers, bone cancers, brain cancers, breast cancers (e.g., triplenegative breast cancer), cervical cancers, colorectal cancers,endometrial cancers, esophageal cancers, eye cancers, gastric cancers,head and neck cancers, kidney cancers (e.g., advanced renal cellcarcinoma), liver cancers (e.g., hepatocellular carcinoma,cholangiocarcinoma), lung cancers (e.g., non-small cell lung cancer,mesothelioma, small cell lung cancer), head and neck cancers, melanomas(e.g., unresectable or metastatic melanoma, advanced malignantmelanoma), oral cancers, ovarian cancers, penile cancers, prostatecancers, pancreatic cancers, skin cancers (e.g., Merkel cell carcinoma),testicular cancers, thyroid cancers, uterine cancers, vaginal cancers,and tumors with evidence of DNA mismatch repair deficiency. The cancersmay be comprised of tumor cells that express PD-L1 or PD-L2, cancerscomprised of tumor cells that do not express PD-L1 or PD-L2, or cancerscomprised of tumor cells, some of which express PD-L1 or PD-L2 and someof which do not. The cancer may be newly diagnosed and naïve totreatment, or may be relapsed, refractory, or relapsed and refractory,or a metastatic form of a solid tumor. In some embodiments, the solidtumor is selected from bladder cancer, breast cancer, head and neckcancer, kidney cancer, lung cancer, lymphoma, melanoma, and gastriccancer. In some embodiments, the solid tumor is selected from: melanoma(e.g., unresectable or metastatic melanoma), lung cancer (e.g.,non-small cell lung cancer), and renal cell carcinoma (e.g., advancedrenal cell carcinoma). In some embodiments, the solid tumor is selectedfrom triple negative breast cancer, ovarian cancer, hepatocellularcarcinoma, gastric cancer, small cell lung cancer, mesothelioma,cholangiocarcinoma, Merkel cell carcinoma and tumors with evidence ofDNA mismatch repair deficiency. In certain embodiments, the melanoma isBRAF V600 wild-type unresectable or metastatic melanoma. In othercertain embodiments, the melanoma is BRAF V600 mutation-positiveunresectable or metastatic melanoma. In certain embodiments, the lungcancer is metastatic non-small cell lung cancer with progression on orafter platinum-based chemotherapy. In certain embodiments, the lungcancer is locally advanced or metastatic non-small cell lung cancer thathas failed platinum-based therapy and is naïve to a PD-1 targetingagent. In certain embodiments, the head and neck cancer is metastatic(disseminated) head and neck squamous cell carcinoma of the oral cavity,oropharynx, hypopharynx, and larynx that is considered incurable bylocal therapies. In certain embodiments, the renal cell carcinoma isadvanced renal cell carcinoma that has received prior anti-angiogenictherapy.

In some embodiments, the method of the disclosure involves administeringto a human patient having a blood malignancy an amount of an anti-PD-1antibody that antagonizes PD-1, and kills malignant cells at a rateeffective to provide therapeutic benefit. The cancers may be comprisedof malignant cells that express PD-L1 or PD-L2, cancers comprised ofmalignant cells that do not express PD-L1 or PD-L2, or cancers comprisedof malignant cells, some of which express PD-L1 or PD-L2 and some ofwhich do not. The cancer may be newly diagnosed and naïve to treatment,or may be relapsed, refractory, or relapsed and refractory, or ametastatic form of a blood malignancy. Blood-borne malignancies that maybe treated with the anti-PD-1 antibody include, but are not limited to,myelomas (e.g., multiple myeloma), lymphomas (e.g., Hodgkin's lymphoma,non-Hodgkin's lymphoma, Waldenström's macroglobulinemia, mantle celllymphoma), leukemias (e.g., chronic lymphocytic leukemia, acute myeloidleukemia, acute lymphocytic leukemia), and myelodysplastic syndromes.

As discussed above, the presently disclosed anti-PD-1 antibodiesmodulate an immunological response. Accordingly, patients havingcompromised immune systems may be excluded from treatment. In someembodiments, a patient is excluded after meeting one or more of thefollowing criteria: (1) Active or prior documented autoimmune disease(including, but not limited to, inflammatory bowel disease, celiacdisease, Wegener syndrome) within the past 2 years. (Subjects withchildhood atopy or asthma, vitiligo, alopecia, Hashimoto syndrome,Grave's disease, or psoriasis not requiring systemic treatment (withinthe past 2 years) are not excluded); (2) History of primaryimmunodeficiency, bone marrow transplantation, chronic lymphocyticleukemia, solid organ transplantation, or previous clinical diagnosis oftuberculosis; (3) History of a coagulopathy or a platelet disorder; (4)Confirmed positive test results for human immunodeficiency virus (HIV),or subjects with chronic or active hepatitis B or C. (Subjects who havea history of hepatitis B or C who have documented cures after anti-viraltherapy may be enrolled); (5) Prior grade ≥3 immune-mediatedneurotoxicity or pneumonitis while receiving immunotherapy (includingbut not limited to agents directed against CTLA-4, PD-L1, or PD-1). Inaddition, any other prior grade ≥3 immune-mediated adverse event whilereceiving immunotherapy that has not resolved or become asymptomaticwithin 3 months; (6) Receipt of live, attenuated vaccine within 28 daysprior to the first dose of the anti-PD-1 antibody.

An anti-PD-1 antibody of the disclosure may be administered alone(monotherapy) or adjunctive to, or with, other anti-cancer therapiesand/or targeted or non-targeted anti-cancer agents. When administered asan anti-PD-1 monotherapy, one or more antibodies may be used. Whetheradministered as monotherapy or adjunctive to, or with, other therapiesor agents, an amount of anti-PD-1 antibody is administered such that theoverall treatment regimen provides therapeutic benefit.

By therapeutic benefit is meant that the use of anti-PD-1 antibodies totreat cancer in a patient results in any demonstrated clinical benefitcompared with no therapy (when appropriate) or to a known standard ofcare. Clinical benefit can be assessed by any method known to one ofordinary skill in the art. In one embodiment, clinical benefit isassessed based on objective response rate (ORR) (determined using RECISTversion 1.1), duration of response (DOR), progression-free survival(PFS), and/or overall survival (OS). In some embodiments, a completeresponse indicates therapeutic benefit. In some embodiments, a partialresponse indicates therapeutic benefit. In some embodiments, stabledisease indicates therapeutic benefit. In some embodiments, an increasein overall survival indicates therapeutic benefit. In some embodiments,therapeutic benefit may constitute an improvement in time to diseaseprogression and/or an improvement in symptoms or quality of life. Inother embodiments, therapeutic benefit may not translate to an increasedperiod of disease control, but rather a markedly reduced symptom burdenresulting in improved quality of life. As will be apparent to those ofskill in the art, a therapeutic benefit may be observed using theanti-PD-1 antibodies alone (monotherapy) or adjunctive to, or with,other anti-cancer therapies and/or targeted or non-targeted anti-canceragents.

Typically, therapeutic benefit is assessed using standard clinical testsdesigned to measure the response to a new treatment for cancer. Toassess the therapeutic benefits of the anti-PD-1 antibodies describedherein one or a combination of the following tests can be used: (1) theResponse Evaluation Criteria In Solid Tumors (RECIST) version 1.1, (2)immune-related RECIST (irRECIST), (3) the Eastern Cooperative OncologyGroup (ECOG) Performance Status, (4) immune-related response criteria(irRC), (5) disease evaluable by assessment of tumor antigens, (6)validated patient reported outcome scales, and/or (7) Kaplan-Meierestimates for overall survival and progression free survival.

Assessment of the change in tumor burden is an important feature of theclinical evaluation of cancer therapeutics. Both tumor shrinkage(objective response) and time to the development of disease progressionare important endpoints in cancer clinical trials. Standardized responsecriteria, known as RECIST (Response Evaluation Criteria in SolidTumors), were published in 2000. An update (RECIST 1.1) was released in2009. RECIST criteria are typically used in clinical trials whereobjective response is the primary study endpoint, as well as in trialswhere assessment of stable disease, tumor progression or time toprogression analyses are undertaken because these outcome measures arebased on an assessment of anatomical tumor burden and its change overthe course of the trial. TABLE 4 provides the definitions of theresponse criteria used to determine objective tumor response to a studydrug, such as the anti-PD-1 antibodies described herein.

TABLE 4 Response Criteria Complete Response Disappearance of all targetlesions. Any pathological lymph nodes (CR) (whether target ornon-target) must have reduction in short axis to <10 mm. PartialResponse At least a 30% decrease in the sum of diameters of targetlesions, taking as (PR) reference the baseline sum diameters.Progressive Disease At least a 20% increase in the sum of diameters oftarget lesions, taking as (PD) reference the smallest sum on study (thisincludes the baseline sum if that is the smallest on study). In additionto the relative increase of 20%, the sum must also demonstrate anabsolute increase of at least 5 mm. (Note: the appearance of one or morenew lesions is also considered progression). Stable Disease Neithersufficient shrinkage to qualify for PR nor sufficient increase to (SD)qualify for PD, taking as reference the smallest sum diameters while onstudy.

Secondary outcome measures that can be used to determine the therapeuticbenefit of the anti-PD-1 antibodies described herein include, ObjectiveResponse Rate (ORR), Progression Free Survival (PFS), Overall Survival(OS), Duration of Overall Response (DOR), and Depth of Response (DpR).ORR is defined as the proportion of the participants who achieve acomplete response (CR) or partial response (PR). PFS is defined as thetime from the first dose date of an anti-PD-1 antibody to either diseaseprogression or death, whichever occurs first. OS is defined as thelength of time from either the date of diagnosis or the start oftreatment for a disease, that patients diagnosed with the disease arestill alive. DOR is defined as the time from the participant's initialCR or PR to the time of disease progression. DpR is defined as thepercentage of tumor shrinkage observed at the maximal response pointcompared to baseline tumor load. Clinical endpoints for both ORR and PFScan be determined based on RECIST 1.1 criteria described above.

Additional criteria that may be used for clinical evaluation specific tocancer patients undergoing immune therapy treatment include thestandardized immune-related RECIST (irRECIST) criteria. See, e.g.,Nishino, M. et al. Eur. J. Radiol., 84(7), pages 1259-1268 (2015 July).These guidelines modified the RECIST 1.1 criteria above withconsideration of potential immunomodulatory effects. TABLE 5 providesthe definitions of the response criteria used to determine objectivetumor response to an immunomodulatory drug, such as the anti-PD-1antibodies described herein.

TABLE 5 Response Criteria Complete Response Complete disappearance ofall measurable and non-measurable lesions. (irCR) Lymph nodes mustdecrease to <10 mm in short axis. Partial Response Decrease of ≥30% intotal measured tumor burden relative to baseline, (irPR) non-targetlesions are irNN, and no unequivocal progression of new non- measurablelesions Progressive Disease At least a 20% increase and at least 5 mmabsolute increase in TMTB (irPD) compared to nadir, or irPD fornon-target or new non-measurable lesions. Confirmation of progression isrecommended at least 4 weeks after the first irPD assessment. Non-irCRor non- No target disease was identified at baseline and at follow-upthe patient irPD (irNN) fails to meet criteria for irCR or irPD StableDisease Neither sufficient shrinkage to qualify for irPR nor sufficientincrease to (irSD) qualify for irPD, taking as reference the smallestsum diameters while on study. irNE Used in exceptional cases whereinsufficient data exists.

The ECOG Scale of Performance Status shown in TABLE 6 is used todescribe a patient's level of functioning in terms of their ability tocare for themselves, daily activity, and physical ability. The scale wasdeveloped by the Eastern Cooperative Oncology Group (ECOG), now part ofthe ECOG-ACRIN Cancer Research Group, and published in 1982.

TABLE 6 Grade ECOG Performance Status 0 Fully active, able to carry onall pre-disease performance without restriction 1 Restricted inphysically strenuous activity but ambulatory and able to carry out workof a light or sedentary nature, e.g., light house work, office work 2Ambulatory and capable of all selfcare but unable to carry out any workactivities; up and about more than 50% of waking hours 3 Capable of onlylimited selfcare; confined to bed or chair more than 50% of waking hours4 Completely disabled; cannot carry on any selfcare; totally confined tobed or chair 5 Dead

Another set of criteria that can be used to characterize fully and todetermine response to immunotherapeutic agents, such as antibody-basedcancer therapies, is the immune-related response criteria (irRC), whichwas developed for measurement of solid tumors in 2009, and updated in2013 (Wolchok, et al. Clin. Cancer Res. 2009; 15(23): 7412-7420 andNishino, et al. Clin. Cancer Res. 2013; 19(14): 3936-3943). The updatedirRC criteria are typically used to assess the effect of animmunotherapeutic agent, such as an anti-PD-1 antibody described herein,on tumor burden, and defines response according to TABLE 7.

TABLE 7 Response Criteria Complete Response Disappearance of all targetlesions in two consecutive observations not less (CR) than 4 weeks apartPartial Response At least a 30% decrease in the sum of the longestdiameters of target (PR) lesions, taking as reference the baseline sumdiameters. Progressive Disease At least a 20% increase in the sum ofdiameters of target lesions, taking as (PD) reference the smallest sumon study (this includes the baseline sum if that is the smallest onstudy). (Note: the appearance of one or more new lesions is notconsidered progression. The measurement of new lesions is included inthe sum of the measurements). Stable Disease Neither sufficientshrinkage to qualify for PR nor sufficient increase to (SD) qualify forPD, taking as reference the smallest sum diameters while on study.

Tumor antigens that can be used to evaluate the therapeutic benefit ofthe anti-PD-1 antibodies described herein include ApoE, CD11c, CD40,CD45 (PTPRC), CD49D (ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS4A7,PIK3AP1, PIK3CD, CD74, CCL5, CCR5, CXCL10, IFNG, IL10RA1, IL-6, ACTA2,COL7A1, LOX, LRRC15, MCPT8, MMP10, NOG, SERPINE1, STAT1, TGFBR1, CTSS,PGF, VEGFA, C1QA, C1QB, ANGPTL4, EGLN, ANGPTL4, EGLN3, BNIP3, AIF1,CCL5, CXCL10, CXCL11, IFI6, PLOD2, KISS1R, STC2, DDIT4, OX40, OX40L,PFKFB3, PGK1, PDK1, AKR1C1, AKR1C2, CADM1, CDH11, COL6A3, CTGF, HMOX1,KRT33A, LUM, WNT5A, IGFBP3, MMP14, CDCP1, PDGFRA, TCF4, TGF, TGFB1,TGFB2, CD11b, ADGRE1 (EMR1, F4/80), CD86, CD68, MHC-Class II, CD3,HLA-DR, CD4, CD3, CD5, CD19, CD7, CD8, CD16, TCRαβ, TCRγδ, PD-1, PD-L1,CTLA-4, acid phosphatase, ACTH, alkaline phosphatase, alpha-fetoproteinCA-125, CA15-3, CA19-9, CA-195, C-212, CA-549, calcitonin,catecholamines, cathepsin-D, CEA, ERBB2 (HER2/neu), chromagranin-A,c-Myc, EGFR, ERA (estrogen receptor assay), ferritin, gastrin, 5-HIAA,hCG, alpha-HCG, beta-HCG, HVA, LDH1-5, NSE (neuron specific enolase),pancreatic polypeptide, PLAP, PLP, PRA (progesterone receptor A),proinsulin C-peptide, PSA, SMA, SCC, thyroglobulin, TDT, TPA, andalpha-TSH. These tumor antigens can be assessed at the DNA, RNA orprotein level using DNA sequencing techniques, RNA sequencingtechniques, gene chip microarray, PCR based methods, flow cytometry orimmunohistochemistry methods as known to experts in the art.

One exemplary therapeutic benefit resulting from the use of anti-PD-1antibodies described herein to treat solid tumors, whether administeredas monotherapy or adjunctive to, or with, other therapies or agents, isa complete response. Another exemplary therapeutic benefit resultingfrom the use of anti-PD-1 antibodies described herein to treat solidtumors, whether administered as monotherapy or adjunctive to, or with,other therapies or agents, is a partial response.

Validated patient reported outcome scales can also be used to denoteresponse provided by each patient through a specific reporting system.Rather than being disease focused, such outcome scales are concernedwith retained function while managing a chronic condition. Onenon-limiting example of a validated patient reported outcome scale isPROMIS® (Patient Reported Outcomes Measurement Information System) fromthe United States National Institutes of Health. For example, PROMIS®Physical Function Instrument for adult cancer patients can evaluateself-reported capabilities for the functioning of upper extremities(e.g., dexterity), lower extremities (e.g., walking or mobility), andcentral regions (e.g., neck, back mobility), and includes routine dailyactivities, such as running errands.

Kaplan-Meier curves (Kaplan and Meier, J. Am. Stat. Assoc. 1958;53(282): 457-481) can also be used to estimate overall survival andprogression free survival for cancer patients undergoing anti-PD-1antibody therapy in comparison to standard of care.

7.6.2. Adjunctive Therapies

The anti-PD-1 antibodies may be used adjunctive to, or with, otheragents or treatments having anti-cancer properties. When usedadjunctively, the anti-PD-1 antibody and other agent(s) may beformulated together in a single, combination pharmaceutical formulation,or may be formulated and administered separately, either on a singlecoordinated dosing regimen or on different dosing regimens. Agentsadministered adjunctive to or with the anti-PD-1 antibodies willtypically have complementary activities to the anti-PD-1 antibodies suchthat the antibodies and other agents do not adversely affect each other.

Agents that may be used adjunctively with anti-PD-1 antibodies include,but are not limited to, alkylating agents, angiogenesis inhibitors,antibodies, antimetabolites, antimitotics, antiproliferatives,antivirals, aurora kinase inhibitors, apoptosis promoters (for example,Bcl-2 family inhibitors), activators of death receptor pathway, Bcr-Ablkinase inhibitors, BiTE (Bi-Specific T cell Engager) antibodies,antibody drug conjugates, biologic response modifiers, Bruton's tyrosinekinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycleinhibitors, cyclooxygenase-2 inhibitors, DVDs, leukemia viral oncogenehomolog (ErbB2) receptor inhibitors, growth factor inhibitors, heatshock protein (HSP)-90 inhibitors, histone deacetylase (HDAC)inhibitors, hormonal therapies, immunologicals, inhibitors of inhibitorsof apoptosis proteins (IAPs), intercalating antibiotics, kinaseinhibitors, kinesin inhibitors, Jak2 inhibitors, mammalian target ofrapamycin inhibitors, microRNAs, mitogen-activated extracellularsignal-regulated kinase inhibitors, multivalent binding proteins,non-steroidal anti-inflammatory drugs (NSAIDs), poly ADP (adenosinediphosphate)-ribose polymerase (PARP) inhibitors, platinumchemotherapeutics, polo-like kinase (Plk) inhibitors, phosphoinositide-3kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs,pyrimidine analogs, receptor tyrosine kinase inhibitors,retinoids/deltoids plant alkaloids, small inhibitory ribonucleic acids(siRNAs), topoisomerase inhibitors, ubiquitin ligase inhibitors, and thelike, as well as combinations of one or more of these agents.

BiTE antibodies are bispecific antibodies that direct T-cells to attackcancer cells by simultaneously binding the two cells. The T-cell thenattacks the target cancer cell. Examples of BiTE antibodies includeadecatumumab (Micromet MT201), blinatumomab (Micromet MT103) and thelike. Without being limited by theory, one of the mechanisms by whichT-cells elicit apoptosis of the target cancer cell is by exocytosis ofcytolytic granule components, which include perforin and granzyme B.

SiRNAs are molecules having endogenous RNA bases or chemically modifiednucleotides. The modifications do not abolish cellular activity, butrather impart increased stability and/or increased cellular potency.Examples of chemical modifications include phosphorothioate groups,2′-deoxynucleotide, 2′-OCH₃-containing ribonucleotides,2′-F-ribonucleotides, 2′-methoxyethyl ribonucleotides, combinationsthereof and the like. The siRNA can have varying lengths (e.g., 10-200bps) and structures (e.g., hairpins, single/double strands, bulges,nicks/gaps, mismatches) and are processed in cells to provide activegene silencing. A double-stranded siRNA (dsRNA) can have the same numberof nucleotides on each strand (blunt ends) or asymmetric ends(overhangs). The overhang of 1-2 nucleotides can be present on the senseand/or the antisense strand, as well as present on the 5′- and/or the3′-ends of a given strand.

Multivalent binding proteins are binding proteins comprising two or moreantigen binding sites. Multivalent binding proteins are engineered tohave the three or more antigen binding sites and are generally notnaturally occurring antibodies. The term “multispecific binding protein”means a binding protein capable of binding two or more related orunrelated targets. Dual variable domain (DVD) binding proteins aretetravalent or multivalent binding proteins binding proteins comprisingtwo or more antigen binding sites. Such DVDs may be monospecific (i.e.,capable of binding one antigen) or multispecific (i.e., capable ofbinding two or more antigens). DVD binding proteins comprising two heavychain DVD polypeptides and two light chain DVD polypeptides are referredto as DVD Ig's. Each half of a DVD Ig comprises a heavy chain DVDpolypeptide, a light chain DVD polypeptide, and two antigen bindingsites. Each binding site comprises a heavy chain variable domain and alight chain variable domain with a total of 6 CDRs involved in antigenbinding per antigen binding site.

Alkylating agents include, but are not limited to, altretamine, AMD-473,AP-5280, apaziquone, bendamustine, brostallicin, busulfan, carboquone,carmustine (BCNU), chlorambucil, CLORETAZINE® (laromustine, VNP 40101M),cyclophosphamide, dacarbazine, estramustine, fotemustine, glufosfamide,ifosfamide, KW-2170, lomustine (CCNU), mafosfamide, melphalan,mitobronitol, mitolactol, nimustine, nitrogen mustard N-oxide,ranimustine, temozolomide, thiotepa, TREANDA® (bendamustine),treosulfan, and trofosfamide.

Angiogenesis inhibitors include, but are not limited to,endothelial-specific receptor tyrosine kinase (Tie-2) inhibitors,epidermal growth factor receptor (EGFR) inhibitors, vascular endothelialgrowth factor receptor (VEGF) inhibitors, delta-like ligand 4 (DLL4)inhibitors, insulin growth factor-2 receptor (IGFR-2) inhibitors, matrixmetalloproteinase-2 (MMP-2) inhibitors, matrix metalloproteinase-9(MMP-9) inhibitors, platelet-derived growth factor receptor (PDGFR)inhibitors, thrombospondin analogs, and vascular endothelial growthfactor receptor tyrosine kinase (VEGFR) inhibitors.

Antibody drug conjugates include, but are not limited to, those thattarget c-Met kinase (e.g., ADCs described in U.S. Pat. No. 7,615,529),LRRC15, CD30 (e.g., ADCETRIS® (brentuximab vedotin)), CS1 (e.g., ADCsdescribed in US publication no. 20160122430), DLL3 (e.g., rovalpituzumabtesirine (ROVA-T)), HER2 (e.g., KADCYLA® (trastuzumab emtansine)), EGFR(e.g., ADCs described in US publication no. 20150337042), and prolactinreceptor (e.g., ADCs described in US publication no. 20140227294).

Antimetabolites include, but are not limited to, ALIMTA® (pemetrexeddisodium, LY231514, MTA), 5-azacitidine, XELODA® (capecitabine),carmofur, LEUSTAT® (cladribine), clofarabine, cytarabine, cytarabineocfosfate, cytosine arabinoside, decitabine, deferoxamine,doxifluridine, eflornithine, EICAR(5-ethynyl-1-β-D-ribofuranosylimidazole-4-carboxamide), enocitabine,ethnylcytidine, fludarabine, 5-fluorouracil alone or in combination withleucovorin, GEMZAR® (gemcitabine), hydroxyurea, ALKERAN® (melphalan),mercaptopurine, 6-mercaptopurine riboside, methotrexate, mycophenolicacid, nelarabine, nolatrexed, ocfosfate, pelitrexol, pentostatin,raltitrexed, ribavirin, triapine, trimetrexate, S-1, tiazofurin,tegafur, TS-1, vidarabine, and UFT.

Antivirals include, but are not limited to, ritonavir, acyclovir,cidofovir, ganciclovir, foscarnet, zidovudine, ribavirin, andhydroxychloroquine.

Aurora kinase inhibitors include, but are not limited to, ABT-348,AZD-1152, MLN-8054, VX-680, Aurora A-specific kinase inhibitors, AuroraB-specific kinase inhibitors and pan-Aurora kinase inhibitors.

Bcl-2 protein inhibitors include, but are not limited to, ABT-263(navitoclax), AT-101 ((−)gossypol), GENASENSE® (G3139 or oblimersen(Bcl-2-targeting antisense oligonucleotide)), IPI-194, IPI-565,N-(4-(4-((4′-chloro(1,1′-biphenyl)-2-yl)methyl)piperazin-1-yl)benzoyl)-4-(((1R)-3-(dimethylamino)-1-((phenylsulfanyl)methyl)propyl)amino)-3-nitrobenzenesulfonamide),N-(4-(4-((2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohex-1-en-1-yl)methyl)piperazin-1-yl)benzoyl)-4-(((1R)-3-(morpholin-4-yl)-1-((phenylsulfanyl)methyl)propyl)amino)-3-((trifluoromethyl)sulfonyl)benzenesulfonamide, venetoclax and GX-070 (obatoclax).

Bcr-Abl kinase inhibitors include, but are not limited to, DASATINIB®(BMS-354825) and GLEEVEC® (imatinib).

BTK inhibitors include, but are not limited to, ibrutinib andacalabrutinib.

CDK inhibitors include, but are not limited to, AZD-5438, BMI-1040,BMS-032, BMS-387, CVT-2584, flavopyridol, GPC-286199, MCS-5A, PD0332991,PHA-690509, seliciclib (CYC-202, R-roscovitine), and ZK-304709.

COX-2 inhibitors include, but are not limited to, ABT-963, ARCOXIA®(etoricoxib), BEXTRA® (valdecoxib), BMS347070, CELEBREX® (celecoxib),COX-189 (lumiracoxib), CT-3, DERAMAXX® (deracoxib), JTE-522,4-methyl-2-(3,4-dimethylphenyl)-1-(4-sulfamoylphenyl-1H-pyrrole), MK-663(etoricoxib), NS-398, parecoxib, RS-57067, SC-58125, SD-8381, SVT-2016,S-2474, T-614, and VIOXX® (rofecoxib).

EGFR inhibitors include, but are not limited to, ABX-EGF, anti-EGFRimmunoliposomes, EGF-vaccine, EMD-7200, ERBITUX® (cetuximab), HR3, IgAantibodies, IRESSA® (gefitinib), TARCEVA® (erlotinib or OSI-774),TAGRISSO® (osimertinib), TP-38, EGFR fusion protein, and TYKERB®(lapatinib).

ErbB2 receptor inhibitors include, but are not limited to, CP-724-714,CI-1033 (canertinib), HERCEPTIN® (trastuzumab), TYKERB® (lapatinib),OMNITARG® (2C4, pertuzumab), TAK-165, GW-572016 (ionafarnib), GW-282974,EKB-569, PI-166, dHER2 (HER2 vaccine), APC-8024 (HER-2 vaccine),anti-HER/2neu bispecific antibody, B7.her2IgG₃, AS HER2 trifunctionalbispecific antibodies, mAB AR-209, and mAB 2B-1.

Histone deacetylase inhibitors include, but are not limited to,depsipeptide, LAQ-824, MS-275, trapoxin, suberoylanilide hydroxamic acid(SAHA), TSA, and valproic acid.

HSP-90 inhibitors include, but are not limited to, 17-AAG-nab, 17-AAG,CNF-101, CNF-1010, CNF-2024, 17-DMAG, geldanamycin, IPI-504, KOS-953,MYCOGRAB® (human recombinant antibody to HSP-90), NCS-683664, PU24FC1,PU-3, radicicol, SNX-2112, STA-9090, and VER49009.

Inhibitors of apoptosis proteins include, but are not limited to,HGS1029, GDC-0145, GDC-0152, LCL-161, and LBW-242.

Activators of death receptor pathway include, but are not limited to,TRAIL, antibodies or other agents that target TRAIL or death receptors(e.g., DR4 and DR5) such as Apomab, conatumumab, ETR2-ST01, GDC0145(lexatumumab), HGS-1029, LBY-135, PRO-1762 and trastuzumab.

Kinesin inhibitors include, but are not limited to, Eg5 inhibitors suchas AZD4877, ARRY-520; and CENPE inhibitors such as GSK923295A.

JAK-2 inhibitors include, but are not limited to, CEP-701 (lesaurtinib),XL019 and INCB018424.

MEK inhibitors include, but are not limited to, ARRY-142886,ARRY-438162, PD-325901, and PD-98059.

mTOR inhibitors include, but are not limited to, AP-23573, CCI-779,everolimus, RAD-001, rapamycin, temsirolimus, ATP-competitiveTORC1/TORC2 inhibitors, including PI-103, PP242, PP30, and Torin 1.

Non-steroidal anti-inflammatory drugs include, but are not limited to,AMIGESIC® (salsalate), DOLOBID® (diflunisal), MOTRIN® (ibuprofen),ORUDIS® (ketoprofen), RELAFEN® (nabumetone), FELDENE® (piroxicam),ibuprofen cream, ALEVE® (naproxen) and NAPROSYN® (naproxen), VOLTAREN®(diclofenac), INDOCIN® (indomethacin), CLINORIL® (sulindac), TOLECTIN®(tolmetin), LODINE® (etodolac), TORADOL® (ketorolac), and DAYPRO®(oxaprozin).

PDGFR inhibitors include, but are not limited to, C-451, CP-673 andCP-868596.

Platinum chemotherapeutics include, but are not limited to, cisplatin,ELOXATIN® (oxaliplatin) eptaplatin, lobaplatin, nedaplatin, PARAPLATIN®(carboplatin), satraplatin, and picoplatin.

Polo-like kinase inhibitors include, but are not limited to, BI-2536.

Phosphoinositide-3 kinase (PI3K) inhibitors include, but are not limitedto, wortmannin, LY294002, XL-147, CAL-120, ONC-21, AEZS-127, ETP-45658,PX-866, GDC-0941, BGT226, BEZ235, and XL765.

Thrombospondin analogs include, but are not limited to, ABT-510,ABT-567, ABT-898, and TSP-1.

VEGFR inhibitors include, but are not limited to, ABT-869, AEE-788,ANGIOZYME™ (a ribozyme that inhibits angiogenesis (RibozymePharmaceuticals (Boulder, Colo.) and Chiron (Emeryville, Calif.)),axitinib (AG-13736), AZD-2171, CP-547,632, CYRAMZA® (ramucirumab),IM-862, MACUGEN® (pegaptamib), NEXAVAR® (sorafenib, BAY43-9006),pazopanib (GW-786034), vatalanib (PTK-787, ZK-222584), SUTENT®(sunitinib, SU-11248), STIVARGA® (regorafenib), VEGF trap, and ZACTIMA™(vandetanib, ZD-6474).

Antibiotics include, but are not limited to, intercalating antibioticsaclarubicin, actinomycin D, amrubicin, annamycin, adriamycin, BLENOXANE®(bleomycin), daunorubicin, CAELYX® or MYOCET® (liposomal doxorubicin),elsamitrucin, epirbucin, glarbuicin, ZAVEDOS® (idarubicin), mitomycin C,nemorubicin, neocarzinostatin, peplomycin, pirarubicin, rebeccamycin,stimalamer, streptozocin, VALSTAR® (valrubicin), and zinostatin.

Topoisomerase inhibitors include, but are not limited to, aclarubicin,9-aminocamptothecin, amonafide, amsacrine, becatecarin, belotecan,BN-80915, CAMPTOSAR® (irinotecan hydrochloride), camptothecin,CARDIOXANE® (dexrazoxine), diflomotecan, edotecarin, ELLENCE® orPHARMORUBICIN® (epirubicin), etoposide, exatecan,10-hydroxycamptothecin, gimatecan, lurtotecan, mitoxantrone, Onivyde™(liposomal irinotecan), orathecin, pirarbucin, pixantrone, rubitecan,sobuzoxane, SN-38, tafluposide, and topotecan.

Antibodies include, but are not limited to, AVASTIN® (bevacizumab),CD40-specific antibodies, chTNT-1/B, denosumab, ERBITUX® (cetuximab),HUMAX-CD4® (zanolimumab), IGF1R-specific antibodies, lintuzumab, OX-40specific antibodies, PANOREX® (edrecolomab), RENCAREX® (WX G250),RITUXAN® (rituximab), ticilimumab, trastuzumab, pertuzumab, VECTIBIX®(panitumumab) and CD20 antibodies types I and II.

Hormonal therapies include, but are not limited to, ARIMIDEX®(anastrozole), AROMASIN® (exemestane), arzoxifene, CASODEX®(bicalutamide), CETROTIDE® (cetrorelix), degarelix, deslorelin, DESOPAN®(trilostane), dexamethasone, DROGENIL® (flutamide), EVISTA®(raloxifene), AFEMA™ (fadrozole), FARESTON® (toremifene), FASLODEX®(fulvestrant), FEMARA® (letrozole), formestane, glucocorticoids,HECTOROL® (doxercalciferol), RENAGEL® (sevelamer carbonate),lasofoxifene, leuprolide acetate, MEGACE® (megesterol), MIFEPREX®(mifepristone), NILANDRON™ (nilutamide), NOLVADEX® (tamoxifen citrate),PLENAXIS™ (abarelix), prednisone, PROPECIA® (finasteride), rilostane,SUPREFACT® (buserelin), TRELSTAR® (luteinizing hormone releasing hormone(LHRH)), VANTAS® (Histrelin implant), VETORYL® (trilostane ormodrastane), and ZOLADEX® (fosrelin, goserelin).

Deltoids and retinoids include, but are not limited to, seocalcitol(EB1089, CB1093), lexacalcitrol (KH1060), fenretinide, PANRETIN®(aliretinoin), ATRAGEN® (liposomal tretinoin), TARGRETIN® (bexarotene),and LGD-1550.

PARP inhibitors include, but are not limited to, ABT-888 (veliparib),KU-59436, AZD-2281 (olaparib), AG-014699 (rucaparib), MK4827(niraparib), BMN-673 (talazoparib), iniparib, BSI-201, BGP-15, INO-1001,and ONO-2231.

Plant alkaloids include, but are not limited to, vincristine,vinblastine, vindesine, and vinorelbine.

Proteasome inhibitors include, but are not limited to, VELCADE®(bortezomib), KYPROLIS® (carfilzomib), MG132, NPI-0052, and PR-171.

Examples of immunologicals include, but are not limited to, interferons,immune checkpoint inhibitors, co-stimulatory agents, and otherimmune-enhancing agents. Interferons include interferon alpha,interferon alpha-2a, interferon alpha-2b, interferon beta, interferongamma-1a, ACTIMMUNE® (interferon gamma-1b) or interferon gamma-n1,combinations thereof and the like. Immune check point inhibitors includeantibodies that target PD-L1 (e.g., durvalumab, atezolizumab, avelumab,MEDI4736, MSB0010718C and MPDL3280A), and CTLA4 (cytotoxic lymphocyteantigen 4; e.g., ipilimumab, tremelimumab). Co-stimulatory agentsinclude, but are not limited to, antibodies against CD3, CD40, CD40L,CD27, CD28, CSF1R, CD137 (e.g., urelumab), B7H1, GITR, ICOS, CD80, CD86,OX40, OX40L, CD70, HLA-DR, LIGHT, LIGHT-R, TIM3, A2AR, NKG2A, KIR (e.g.,lirilumab), TGF-β (e.g., fresolimumab) and combinations thereof.

Other agents include, but are not limited to, ALFAFERONE® (IFN-α),BAM-002 (oxidized glutathione), BEROMUN® (tasonermin), BEXXAR®(tositumomab), CAMPATH® (alemtuzumab), dacarbazine, denileukin,epratuzumab, GRANOCYTE® (lenograstim), lentinan, leukocyte alphainterferon, imiquimod, melanoma vaccine, mitumomab, molgramostim,MYLOTARG™ (gemtuzumab ozogamicin), NEUPOGEN® (filgrastim), OncoVAC-CL,OVAREX® (oregovomab), pemtumomab (Y-muHMFG1), PROVENGE® (sipuleucel-T),sargaramostim, sizofilan, teceleukin, THERACYS® (BacillusCalmette-Guerin), ubenimex, VIRULIZIN® (immunotherapeutic, LorusPharmaceuticals), Z-100 (Specific Substance of Maruyama (SSM)), WF-10(Tetrachlorodecaoxide (TCDO)), PROLEUKIN® (aldesleukin), ZADAXIN®(thymalfasin), ZINBRYTA® (daclizumab high-yield process), and ZEVALIN®(⁹⁰Y-Ibritumomab tiuxetan).

Biological response modifiers are agents that modify defense mechanismsof living organisms or biological responses, such as survival, growth ordifferentiation of tissue cells to direct them to have anti-tumoractivity and include, but are not limited to, krestin, lentinan,sizofiran, picibanil PF-3512676 (CpG-8954), and ubenimex.

Pyrimidine analogs include, but are not limited to, cytarabine (ara C orArabinoside C), cytosine arabinoside, doxifluridine, FLUDARA®(fludarabine), 5-FU (5-fluorouracil), floxuridine, GEMZAR®(gemcitabine), TOMUDEX® (ratitrexed), and TROXATYL™ (triacetyluridinetroxacitabine).

Purine analogs include, but are not limited to, LANVIS® (thioguanine)and PURINETHOL® (mercaptopurine).

Antimitotic agents include, but are not limited to, batabulin,epothilone D (KOS-862),N-(2-((4-hydroxyphenyl)amino)pyridin-3-yl)-4-methoxybenzenesulfonamide,ixabepilone (BMS 247550), TAXOL® (paclitaxel), TAXOTERE® (docetaxel),PNU100940 (109881), patupilone, XRP-9881 (larotaxel), vinflunine, andZK-EPO (synthetic epothilone).

Ubiquitin ligase inhibitors include, but are not limited to, MDM2inhibitors, such as nutlins, and NEDD8 inhibitors such as MLN4924.

Anti-PD-1 antibodies may also be used to enhance the efficacy ofradiation therapy. Examples of radiation therapy include external beamradiation therapy, internal radiation therapy (i.e., brachytherapy) andsystemic radiation therapy.

Anti-PD-1 antibodies may be administered adjunctive to or with otherchemotherapeutic agents such as ABRAXANE™ (ABI-007), ABT-100 (farnesyltransferase inhibitor), ADVEXIN® (Ad5CMV-p53 vaccine), ALTOCOR® orMEVACOR® (lovastatin), AMPLIGEN® (poly I:poly C12U, a synthetic RNA),APTOSYN® (exisulind), AREDIA® (pamidronic acid), arglabin,L-asparaginase, atamestane (1-methyl-3,17-dione-androsta-1,4-diene),AVAGE® (tazarotene), AVE-8062 (combreastatin derivative) BEC2(mitumomab), cachectin or cachexin (tumor necrosis factor), canvaxin(vaccine), CEAVAC® (cancer vaccine), CELEUK® (celmoleukin), CEPLENE®(histamine dihydrochloride), CERVARIX® (human papillomavirus vaccine),CHOP® (C: CYTOXAN® (cyclophosphamide); H: ADRIAMYCIN®(hydroxydoxorubicin); O: Vincristine (ONCOVIN®); P: prednisone), CYPAT™(cyproterone acetate), combrestatin A4P, DAB(389)EGF (catalytic andtranslocation domains of diphtheria toxin fused via a His-Ala linker tohuman epidermal growth factor) or TransMID-107R™ (diphtheria toxins),dacarbazine, dactinomycin, 5,6-dimethylxanthenone-4-acetic acid (DMXAA),eniluracil, EVIZON™ (squalamine lactate), DIMERICINE® (T4N5 liposomelotion), discodermolide, DX-8951f (exatecan mesylate), enzastaurin,EP0906 (epithilone B), GARDASIL® (quadrivalent human papillomavirus(Types 6, 11, 16, 18) recombinant vaccine), GASTRIMMUNE®, GENASENSE®,GMK (ganglioside conjugate vaccine), GVAX® (prostate cancer vaccine),halofuginone, histrelin, hydroxycarbamide, ibandronic acid, IGN-101,IL-13-PE38, IL-13-PE38QQR (cintredekin besudotox), IL-13-pseudomonasexotoxin, interferon-α, interferon-γ, JUNOVAN™ or MEPACT™ (mifamurtide),lonafarnib, 5,10-methylenetetrahydrofolate, miltefosine(hexadecylphosphocholine), NEOVASTAT® (AE-941), NEUTREXIN® (trimetrexateglucuronate), NIPENT® (pentostatin), ONCONASE® (a ribonuclease enzyme),ONCOPHAGE® (melanoma vaccine treatment), ONCOVAX® (IL-2 Vaccine),ORATHECIN™ (rubitecan), OSIDEM® (antibody-based cell drug), OVAREX® MAb(murine monoclonal antibody), paclitaxel, PANDIMEX™ (aglycone saponinsfrom ginseng comprising 20(S)protopanaxadiol (aPPD) and20(S)protopanaxatriol (aPPT)), panitumumab, PANVAC®-VF (investigationalcancer vaccine), pegaspargase, PEG Interferon A, phenoxodiol,procarbazine, rebimastat, REMOVAB® (catumaxomab), REVLIMID®(lenalidomide), RSR13 (efaproxiral), SOMATULINE® LA (lanreotide),SORIATANE® (acitretin), staurosporine (Streptomyces staurospores),talabostat (PT100), TARGRETIN® (bexarotene), TAXOPREXIN®(DHA-paclitaxel), TELCYTA® (canfosfamide, TLK286), temilifene, TEMODAR®(temozolomide), tesmilifene, thalidomide, THERATOPE® (STn-KLH), thymitaq(2-amino-3,4-dihydro-6-methyl-4-oxo-5-(4-pyridylthio)quinazolinedihydrochloride), TNFERADE™ (adenovector: DNA carrier containing thegene for tumor necrosis factor-α), TRACLEER® or ZAVESCA® (bosentan),tretinoin (Retin-A), tetrandrine, TRISENOX® (arsenic trioxide),VIRULIZIN®, ukrain (derivative of alkaloids from the greater celandineplant), vitaxin (anti-alphavbeta3 antibody), XCYTRIN® (motexafingadolinium), XINLAY™ (atrasentan), XYOTAX™ (paclitaxel poliglumex),YONDELIS® (trabectedin), ZD-6126, ZINECARD® (dexrazoxane), ZOMETA®(zolendronic acid), and zorubicin, as well as combinations of any ofthese agents.

In some embodiments, an anti-PD-1 antibody is administered adjunctive toor with an antibody-drug conjugate targeting c-Met kinase for treatingnon-small cell lung cancer, head and neck cancer, pancreatic cancer,colorectal cancer, or gastric cancer.

In some embodiments, an anti-PD-1 antibody is administered adjunctive toor with an antibody-drug conjugate targeting LRRC15 for treatingnon-small cell lung cancer, head and neck cancer, pancreatic cancer,sarcoma, triple negative breast cancer, or melanoma.

In some embodiments, an anti-PD-1 antibody is administered adjunctive toor with an antibody-drug conjugate targeting EGFR for treatingglioblastoma.

In some embodiments, an anti-PD-1 antibody is administered adjunctive toor with an antibody-drug conjugate targeting CS1 for treating a bloodmalignancy such as multiple myeloma.

In some embodiments, an anti-PD-1 antibody is administered adjunctive toor with an antibody-drug conjugate targeting DLL3 for treating smallcell lung cancer or glioblastoma.

In some embodiments, an anti-PD-1 antibody is administered adjunctive toor with an anti-CD40 protein for treating head and neck cancer, lungcancer (such as adenocarcinoma, non-small cell lung cancer,mesothelioma, small cell lung cancer), melanoma, ovarian cancer orpancreatic cancer.

In some embodiments, an anti-PD-1 antibody is administered adjunctive toor with venetoclax for treating a blood malignancy such as chroniclymphocytic leukemia.

In some embodiments, an anti-PD-1 antibody is administered adjunctive toor with ibrutinib for treating a blood malignancy, such as chroniclymphocytic leukemia, mantle cell lymphoma, or Waldenstrom'smacroglobulinemia, or a solid tumor.

In some embodiments, an anti-PD-1 antibody is administered adjunctive toor with ipilimumab and an antibody-drug conjugate targeting c-Met kinasefor treating non-small cell lung cancer.

In some embodiments, an anti-PD-1 antibody is administered adjunctive toor with ipilimumab and an antibody-drug conjugate targeting LRRC15 fortreating non-small cell lung cancer.

7.7. Dosages and Administration Regimens

The amount of anti-PD-1 antibodies administered will depend upon avariety of factors, including but not limited to, the particular type ofsolid tumor treated, the stage of the solid tumor being treated, themode of administration, the frequency of administration, the desiredtherapeutic benefit, and other parameters such as the age, weight andother characteristics of the patient, etc. Determination of dosageseffective to provide therapeutic benefit for specific modes andfrequency of administration is within the capabilities of those skilledin the art.

Dosages effective to provide therapeutic benefit may be estimatedinitially from in vivo animal models or clinical. Suitable animal modelsfor a wide variety of diseases are known in the art.

The anti-PD-1 antibodies disclosed herein may be administered by anyroute appropriate to the condition to be treated. In some embodiments,the anti-PD-1 antibody is any one of the humanized antibodies listed inTABLE 3. In a specific embodiment, the anti-PD-1 antibody has a heavychain according to SEQ ID NO:51 or SEQ ID NO:52, and a light chainaccording to SEQ ID NO:61. An anti-PD-1 antibody will typically beadministered parenterally, i.e., infusion, subcutaneous, intramuscular,intravenous (IV), intradermal, intrathecal, bolus, intratumoralinjection or epidural ((Shire et al., 2004, J. Pharm. Sciences93(6):1390-1402)). In one embodiment, an anti-PD-1 antibody is providedas a lyophilized powder in a vial. The vials may contain 100 mg, 110 mg,120 mg, 150 mg, 200 mg, 250 mg, 300 mg, or 400 mg of anti-PD-1 antibody.Prior to administration, the lyophilized powder is reconstituted withsterile water for injection (SWFI) or other suitable medium to provide asolution containing 20 mg/mL anti-PD-1 antibody. In some embodiments,the resulting reconstituted solution is further diluted with saline orother suitable medium for infusion and administered via an IV infusiontwice every 7 days, once every 7 days, once every 14 days, once every 21days, once every 28 days, once every 35 days, once every 42 days, onceevery 49 days, or once every 56 days. In some embodiments, for the firstcycle, the infusion occurs over 90 minutes. In some embodiments,subsequent infusions are over 60 minutes.

In some embodiments, the anti-PD-1 antibody is administered as an IVinfusion once every 7 days at 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0mg/kg, 3.0 mg/kg, 4.0 mg/kg, 5.0 mg/kg, 6.0 mg/kg, 8.0 mg/kg, or 10.0mg/kg.

In some embodiments, the anti-PD-1 antibody is administered as an IVinfusion once every 14 days at 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0mg/kg, 3.0 mg/kg, 4.0 mg/kg, 5.0 mg/kg, 6.0 mg/kg, 8.0 mg/kg, or 10.0mg/kg.

In some embodiments, the anti-PD-1 antibody is administered as an IVinfusion once every 21 days at 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0mg/kg, 3.0 mg/kg, 4.0 mg/kg, 5.0 mg/kg, 6.0 mg/kg, 8.0 mg/kg, or 10.0mg/kg.

In some embodiments, the anti-PD-1 antibody is administered as an IVinfusion once every 28 days at 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0mg/kg, 3.0 mg/kg, 4.0 mg/kg, 5.0 mg/kg, 6.0 mg/kg, 8.0 mg/kg, or 10.0mg/kg.

In one exemplary embodiment, an anti-PD-1 antibody is used adjunctive toipilimumab (YERVOY®) to treat non-small cell lung cancer. The anti-PD-1antibody is administered via IV infusion once every 21 days at 1.0 mg/kgor 3.0 mg/kg. Ipilimumab is administered by intravenous infusion at adose of 1 mg/kg once every three weeks for four doses. Subsequent to thelast ipilimumab dose, the anti-PD-1 antibody is administered via IVinfusion once every 14 days at 1.0 mg/kg or 3.0 mg/kg. The adjunctiveanti-PD-1 antibody/ipilimumab therapy is continued until diseaseprogression or no longer tolerated by the patient.

In one exemplary embodiment, an anti-PD-1 antibody is used adjunctive toipilimumab (YERVOY®) to treat non-small cell lung cancer. The anti-PD-1antibody is administered via IV infusion once every 14 days at 1.0 mg/kgor 3.0 mg/kg. Ipilimumab is administered by intravenous infusion at adose of 1 mg/kg once every six weeks for four doses. The adjunctiveanti-PD-1 antibody/ipilimumab therapy is continued until diseaseprogression or no longer tolerated by the patient.

In one exemplary embodiment, an anti-PD-1 antibody is used adjunctive toipilimumab (YERVOY®) to treat non-small cell lung cancer. The anti-PD-1antibody is administered via IV infusion once every 14 days at 1.0 mg/kgor 3.0 mg/kg. Ipilimumab is administered by intravenous infusion at adose of 1 mg/kg once every twelve weeks for four doses. The adjunctiveanti-PD-1 antibody/ipilimumab therapy is continued until diseaseprogression or no longer tolerated by the patient.

When administered adjunctive to or with other agents, such as otherchemotherapeutic agents, the anti-PD-1 antibodies may be administered onthe same schedule as the other agent(s), or on a different schedule.When administered on the same schedule, the anti-PD-1 antibody may beadministered before, after, or concurrently with the other agent. Insome embodiments where an anti-PD-1 antibody is administered adjunctiveto, or with, standards of care, the anti-PD-1 antibody may be initiatedprior to commencement of the standard therapy, for example a day,several days, a week, several weeks, a month, or even several monthsbefore commencement of standard of care therapy. In some embodimentswhere an anti-PD-1 antibody is administered adjunctive to, or with,standards of care, the anti-PD-1 antibody may be initiated aftercommencement of the standard therapy, for example a day, several days, aweek, several weeks, a month, or even several months after commencementof standard of care therapy.

As will be appreciated by those of skill in the art, the recommendeddosages for the various agents described above may need to be adjustedto optimize patient response and maximize therapeutic benefit.

7.8. Exemplary Embodiments

The following are exemplary enumerated embodiments of the presentdisclosure.

1. An anti-PD-1 binding protein which comprises (i) a V_(H) chaincomprising three CDRs; and (ii) a V_(L) chain comprising three CDRs,wherein: V_(H) CDR #1 is GYTFTHYGMN (SEQ ID NO:11); V_(H) CDR #2 isWVNTYTGEPTYADDFKG (SEQ ID NO:12); V_(H) CDR #3 is EGEGLGFGD (SEQ IDNO:13); V_(L) CDR #1 is RSSQSIVHSHGDTYLE (SEQ ID NO:14); V_(L) CDR #2 isKVSNRFS (SEQ ID NO:15); and V_(L) CDR #3 is FQGSHIPVT (SEQ ID NO:16).

2. The anti-PD-1 binding protein of embodiment 1, which comprises aV_(H) chain corresponding in sequence to SEQ ID NO:31; and a V_(L) chaincorresponding in sequence to SEQ ID NO:41.

3. The anti-PD-1 binding protein of embodiment 1, which is humanized.

4. The anti-PD-1 binding protein of embodiment 3, which comprises aV_(H) chain corresponding in sequence to SEQ ID NO:36; and a V_(L) chaincorresponding in sequence to SEQ ID NO:42.

5. An anti-PD-1 binding protein which comprises (i) a V_(H) chaincomprising three CDRs; and (ii) a V_(L) chain comprising three CDRs,wherein: V_(H) CDR #1 is GYTFTHYGMN (SEQ ID NO:11); V_(H) CDR #2 isWVNTYTGEPTYADDFKG (SEQ ID NO:12); V_(H) CDR #3 is EGEGMGFGD (SEQ IDNO:23); V_(L) CDR #1 is RSSQSIVHSHGDTYLE (SEQ ID NO:14); V_(L) CDR #2 isKVSNRFS (SEQ ID NO:15); and V_(L) CDR #3 is FQGSHIPVT (SEQ ID NO:16).

6. The anti-PD-1 binding protein of embodiment 5, which comprises aV_(H) chain corresponding in sequence to SEQ ID NO:33; and a V_(L) chaincorresponding in sequence to SEQ ID NO:42.

7. The anti-PD-1 binding protein of any one of embodiments 1 to 6, whichis an IgG.

8. The anti-PD-1 binding protein of embodiment 7, which is an IgG₁,optionally comprising a variant CH2 domain comprising the amino acidsubstitutions L234A and L235A.

9. The anti-PD-1 binding protein of embodiment 7, which is an IgG₄,optionally comprising a variant Fc region comprising the amino acidsubstitution S228P.

10. The anti-PD-1 binding protein of embodiment 8 which comprises aheavy chain corresponding to the sequence of SEQ ID NO:51 or SEQ IDNO:52, and a light chain corresponding to the sequence of SEQ ID NO:61.

11. The anti-PD-1 binding protein of any one of embodiments 1 to 10which has a K_(D) of less than about 100 nM.

12. The anti-PD-1 binding protein of embodiment 11 which has a K_(D) ofless than about 10 nM.

13. A pharmaceutical composition comprising the anti-PD-1 bindingprotein of any one of embodiments 1 to 12, and a pharmaceuticallyacceptable carrier.

14. A nucleic acid comprising a nucleotide sequence encoding theanti-PD-1 binding protein of any one of embodiments 1 to 12.

15. A vector comprising the nucleic acid of embodiment 14.

16. A prokaryotic host cell transformed with the vector of embodiment15.

17. A eukaryotic host cell transformed with the vector of embodiment 15.

18. A eukaryotic host cell engineered to express the nucleic acid ofembodiment 14.

19. The eukaryotic host cell of embodiment 18 which is a mammalian hostcell.

20. A method of producing an anti-PD-1 binding protein thereof,comprising: (a) culturing the host cell of embodiment 17 or embodiment18 and (b) recovering the anti-PD-1 binding protein.

21. A method of activating the immune system, comprising administeringto a patient in need thereof the anti-PD-1 binding protein of any one ofembodiments 1 to 12, or the pharmaceutical composition according toembodiment 13.

22. A method of treating a cancer, comprising administering to a patientin need thereof the anti-PD-1 binding protein of any one of embodiments1 to 12, or the pharmaceutical composition according to embodiment 13.

23. The method of embodiment 22, wherein the cancer is selected frombladder cancer, breast cancer, head and neck cancer, kidney cancer, lungcancer, lymphoma, melanoma, and stomach cancer.

24. The method of embodiment 23, wherein the lung cancer is non-smallcell lung cancer.

25. The method of embodiment 22, in which the anti-PD-1 binding proteinis administered as a monotherapy.

26. The method of embodiment 22, in which the anti-PD-1 binding proteinis administered adjunctive to or with another agent commonly used totreat the cancer.

27. The method of embodiment 26, in which the other agent is selectedfrom radiation, chemotherapy, an antibody drug conjugate, an anti-CD40antibody, an anti-CTLA-4 antibody and an anti-OX40 antibody.

28. The method of embodiment 27, in which the chemotherapy is cisplatin,carboplatin, paclitaxel, docetaxel, gemcitabine, vinorelbine,vinblastine, irinotecan, etoposide, or pemetrexed, or a pharmaceuticallyacceptable salt thereof.

29. The method of embodiment 27, in which the antibody drug conjugatetargets c-Met kinase.

30. The method of embodiment 27, in which the antibody drug conjugatetargets LRRC15.

31. The method of embodiment 27, in which the antibody drug conjugatetargets EGFR.

32. The method of embodiment 27, in which the antibody drug conjugatetargets CS1.

33. The method of embodiment 27, in which the antibody drug conjugate isrovalpituzumab tesirine.

34. The method of embodiment 27, in which the anti-CTLA-4 antibody isipilimumab.

8. Examples

The following Examples, which highlight certain features and propertiesof embodiments of the anti-PD-1 antibodies described herein are providedfor purposes of illustration, and not limitation.

Example 1: Materials and Methods

8.1.1. Antibody Binding to Plate Bound Human PD-1 by ELISA

Immunolon 4×HB 96 well plates were coated with 1 ug/ml of human PD-1 Fcfusion in at 4° C. overnight. Plates were blocked with PBS containing 1%BSA for 30 minutes at room temperature and then washed three times withPBS containing 0.1% Tween 20 (PBST) using a plate washer. PD-1-coatedplates were then incubated with indicated concentrations of antibodiesat room temperature (RT) for one hour. Plates were washed four timeswith PBST and then incubated for 1 hour at room temperature with 100 μLof goat anti-human Fab fragment specific-biotin prepared to a dilutionof 1:5000 in PBS containing 1% BSA. Plates were washed five times inPBST and 100 μL of a 1:1000 dilution of Streptavidin-HRP was added toeach well and incubated for 30 minutes at RT. Plates were washed fivetimes in PBST and 100 μL of TMB One Component were added to each welland incubated at RT until color developed (approximately 5-10 minutes).Optical density (OD) was read at 650 nm using Spectromax190 (MolecularDevices).

8.1.2. Antibody Binding to Cell Surface Expressed Human PD-1

PD-1 expressing Jurkat cells were harvested from flasks and resuspendedto 2×10⁶ cells/mL in PBS containing 1% BSA. 100 μL of cells were addedto a round bottom 96 well plate containing 100 μL of titrated testantibody or isotype control. Cells were incubated with antibodies atroom temperature for 25 minutes and then washed twice with PBScontaining 1% BSA. The cell pellet was resuspended in 100 μL of a 1:250diluted secondary antibody goat anti-human Fab PE. Following 25 minutesincubation at RT, cells were washed twice with PBS containing 1% BSA andresuspended in 200 μL of 1% BSA. Cells were analyzed using BectonDickinson FACSCanto flow cytometer. Data was analyzed using BD FACSDivasoftware (version 8.0.1).

8.1.3. Antibody Binding to Plate Bound Cynomolgus Monkey PD-1 by ELISA

Immunolon 4×HB 96-well plates were coated with 1 μg/mL of cynomolgusPD-1 Fc fusion in DPBS at 4° C. overnight. Plates were blocked with PBScontaining 1% BSA for 30 minutes at RT and then washed three times withPBST (PBS Tween 20 0.1%) using a plate washer. PD-1-coated plates werethen incubated with indicated concentrations of antibodies at roomtemperature for one hour. Plates were washed four times with PBST andthen incubated for 1 hour at RT with 100 μL of goat anti-human Fabfragment specific biotin prepared to a dilution of 1:5000 in PBScontaining 1% BSA. Plates were washed five times in PBST and 100 μL of a1:1000 dilution of Streptavidin-HRP was added to each well and incubatedfor 30 minutes at RT. Plates were subsequently washed five times in PBSTand 100 μL of TMB. One component was added to each well and incubated atRT until color developed (approximately 5-10 minutes). Optical density(OD) was read at 650 nm using a Spectromax190 (Molecular Devices).

8.1.4. Antibody Binding to Activated Human CD4+ T Cells

Human peripheral blood mononuclear cells (PBMCs) were isolated frombuffy coats purchased from Stanford Blood Center (Palo Alto, Calif.).Briefly, buffy coats were diluted in a 1:1 ratio with PBS withoutmagnesium and calcium. Diluted blood (30 mL) was layered over 15 mL of90% Ficoll-Paque Plus prepared in PBS without magnesium and calciumcontained in SepMate tubes. The tubes were spun at 1200 g for 10minutes. The interphase was collected and washed twice in 1×PBS. PBMCwere cultured at 2×10⁶ cells/mL for 48 hrs in RPMI media containing 10%HI FCS with 1 μg/mL PHA and 50 U/mL recombinant human IL-2. The cellswere collected, washed and incubated with antibodies at RT for 25minutes. Labeled cells were washed twice with PBS containing 1% BSA.Cells were resuspended in 100 μL of PBS+1% BSA and a 1:250 dilution ofPE conjugated goat anti-human Fab fragment and anti-CD4-FITC were added.After 30 minutes, cells were washed twice with PBS containing 1% BSA andresuspended in 200 μL of 1% BSA. Cells were analyzed using BectonDickinson FACSCanto flow cytometer. Data was analyzed using BD FACSDivasoftware (version 8.0.1).

8.1.5. Binding Affinity for PD-1 by Surface Plasmon Resonance

The binding kinetics of anti-PD-1 antibodies for recombinant solublePD-1 ECD (extracellular domain) were determined by surface plasmonresonance-based measurements made on Biacore T200 instrument at 25° C.using an anti-Fc capture assay approach. Recombinant extracellulardomains of human PD-1 (residues 1-167) and cynomolgus PD-1 (residues21-167) were purchased from a commercial source and further purified bygel filtration in 10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA. Chippreparation and binding kinetic measurements were made in the assaybuffer HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween20). For anti-Fc capture chip preparation, approximately 2000 RU of goatanti-human IgG Fc polyclonal antibody diluted to 25 μg/mL in 10 mMsodium acetate (pH 4.5), was directly immobilized across a CMS biosensorchip using a standard amine coupling kit according to manufacturer'sinstructions and procedures. Unreacted moieties on the biosensor surfacewere blocked with ethanolamine. For binding kinetics measurements, eachassay cycle comprises the following steps: (1) capture of test antibodyon test surface only; (2) analyte injection (PD-1 ECD or buffer only)over both reference and test surface, 240 μL at 80 μL/min, after whichthe dissociation was monitored for 900 seconds at 80 μL/min; and (3)regeneration of capture surface by 10 mM glycine hydrochloride, pH 1.5injections over both reference and test surface. During the assay, allmeasurements were referenced against the capture surface alone (i.e.,with no captured test antibody) and buffer-only injections were used fordouble referencing. PD-1 injections ranged in concentration from 900 nMto 11.1 nM in a randomized 3-fold dilution series. Data were processedand fitted globally to a 1:1 binding model using Biacore T200 Evaluationsoftware to determine the binding kinetic rate constants, k_(a)(M⁻¹·sec⁻¹) and k_(d) (sec⁻¹), and the equilibrium dissociation constantK_(D) (M).

8.1.6. Blocking of PD-1 Interaction with PD-L1 and PD-L2

Human PD-1 expressing HEK 293G cells were harvested from confluentflasks. Cells were resuspended in PBS at a concentration of 2×10⁶cells/mL. Cells (1×10⁵) were added to each well of a 96-well V bottomplate and cells were blocked for 15 min at 4° C. using human FcR block.In separate plates, test antibodies and isotype control solutions wereprepared using a 3-fold serial dilution of the 20 μg/mL startingconcentration. Cells were washed in 1×PBS and 50 μL of the preparedantibody dilutions and 50 μL of the PD-L1 His-tagged or PD-L2 His-taggedligands (10 μg/ml) were added to the plate containing cells. Cells wereincubated at 4° C. for 30 minutes and washed twice with 1×PBS. Anti-HisAPC antibody (50 μL) prepared at a dilution of 1:50 in PBS was added toeach well and incubated for 30 minutes at 4° C. The cells were washedtwice, resuspended in PBS and acquired on LSR II Fortessa (BDBiosciences, San Jose, Calif.).

8.1.7. Allogeneic Human Mixed Lymphocyte Reaction (MLR) Assay UsingPurified CD4 T Cells and Dendritic Cells

Human PBMCs were isolated from buffy coats purchased from Stanford BloodCenter (Palo Alto, Calif.). Briefly, buffy coats were diluted in a 1:1ratio with PBS without magnesium and calcium. Diluted blood (30 mL) waslayered over 15 mL of 90% Ficoll-Paque Plus prepared in PBS withoutmagnesium and calcium contained in SepMate tubes. The tubes were spun at1200 g for 10 minutes. The interphase was collected and washed twice in1×PBS. Cells were resuspended at 1×10⁸ per mL in AIM-V media containingbeta mercaptoethanol. Dendritic cells (DCs) were derived by culturingplastic adherent PBMCs in T75 flasks in the presence of 80 ng/mL GM-CSFand 50 ng/mL IL-4 for 7 days. On day 5, 50 pg/mL IL-1α and 200 pg/mLTNF-α was added to the DC cultures. On day 7, DCs were harvested fromflasks, irradiated for 7.3 minutes at 414 R/min, and resuspended to afinal concentration of 1×10⁵ cells/mL in complete media (RPMI with LGlutamine containing 10% FBS, 1× non-essential vitamins, 1% Pen/Strepsolution, 1% sodium pyruvate, 1% HEPES). Allogeneic human CD4 T cellswere isolated using the CD4 T cell isolation kit. DCs (1×10⁴/well) andpurified CD4 T cells (1×10⁵/well) were added to a U bottom plate. Testantibody or isotype control (10 μg/mL) was added to the plate containingDCs and T cells. After five days of incubation, supernatant wascollected and analyzed for IL-2 and IFN-γ using Milliplex cytometricbead array kit. IFN-γ was measured using BioRad Bioplex System (Bioplexmanager 6.0).

8.1.8. Antigen Recall Response to Tetanus Toxoid

Human PBMCs were isolated from buffy coats purchased from Stanford BloodCenter as previously described. Cells were resuspended to a finalconcentration of 2×10⁶ cells/mL in AIM-V media containing betamercaptoethanol. PBMCs (2×10⁵) were used per well with 0.2 μg/mL tetanustoxoid. Antibody was titrated and the plates were incubated for fivedays. Supernatants were collected on day 5 and assessed for IFN-γ usinga Milliplex cytometric bead array kit. IFN-γ was measured using BioRadBioplex System (Bioplex manager 6.0).

Example 2: Generation and Humanization of Mouse Anti-PD-1 Antibodies

Mice were immunized according to the methods known in the art (E.Harlow, D. Lane. Antibody: A Laboratory Manual, (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1998)). Isotype of eachmonoclonal antibody was determined using the Mouse Isotyping kit(Roche). Hybridoma clones producing antibodies of interest were purifiedand further characterized for affinity by surface plasmon resonance andligand competition (ELISA).

Cloning and construction of the expression vector were accomplished bymethods known in the art for expression of recombinant monoclonalantibodies.

Humanization of the antibody V region was carried out as outlined byQueen, C. et al. (Proc. Natl. Acad. Sci. USA, 1989; 86:10029-10033). Thecanonical structures of the CDRs were determined according to Huang etal. (Methods, 2005; 36:35-42). Human variable germline sequences withthe same or most similar CDR canonical structures were identified, andappropriate human V_(H), V_(L), and J segment sequences were selected toprovide the frameworks for the anti-PD-1 variable region. At frameworkpositions in which the computer model suggested significant contact withthe CDRs, the amino acids from the murine anti-PD-1 V regions weresubstituted for the original human framework amino acids(back-mutations). Full amino acid sequences of the V_(H) and V_(L)regions of exemplary mouse and humanized antibodies are shown in FIG. 2.

Anti-PD-1 mouse antibody Mu12A11 was humanized according to the methoddescribed above. The humanized versions of Mu12A11 V_(H) were Hu12A11.1bV_(H) and Hu12A11.2b V_(H). Hu12A11.1b V_(H) had V_(H) 7-4-1 frameworkregions, with four back mutations of V2I, M48V, F67L, and A93T.Hu12A11.2b V_(H) had V_(H) 1-69 framework regions, with seven backmutations of V2I, M48V, V67L, I69F, A71L, E73T, and A93T. Either of thetwo humanized V_(H) could be combined with humanized light chainHu12A11.1a V_(L) which had V_(L) 2-28 framework regions, with one backmutation of I2V.

Example 3: Stability of the Anti-PD-1 Antibodies

Stability of exemplary antibodies were determined by measuring PD-1binding of the antibodies after treatment under oxidative or variabletemperature conditions.

An assessment of liability motifs identified a conserved methionineresidue at Kabat position 99 (M99) that was believed to be both solventexposed and prone to deamidation. Samples of the M99 parent antibodyHu12A11.2b1 were exposed to accelerated degradation conditions toenhance potential deamidation (FIG. 3A). The samples treated with 1%hydrogen peroxide (1% HP) or 1% tert-butyl hydroperoxide (1% TBHP)demonstrated loss of binding affinity, suggesting the potential foroxidation of the methionine-99 residue. A set of antibodies containingpoint mutations at position M99 were constructed, including isoleucine(M991, Hu12A11.2b2 having a V_(H) according to SEQ ID NO:34), valine(M99V, Hu12A11.2b3 having a V_(H) according to SEQ ID NO:35), andleucine (M99L, Hu12A11.2b4 having a V_(H) according to SEQ ID NO:36)mutations. The antibody variants were screened for binding to human PD-1transfected Jurkat cells and EC₅₀ values were determined (FIG. 3B). Allthree point mutation-containing antibodies bound to cell surface PD-1similarly, but the M99L variant antibody showed higher binding activityas compared to M99V or M991 variants.

The M99L variant Hu12A11.2b4 was found to retain fully binding capacityafter temperature stability testing (FIG. 3C). Incubation of Hu12A11.2b4at −80, 5, 25, or 40° C. afforded no significant loss of activity interms of EC₅₀ or maximal fluorescence intensity (MFI).

Example 4: Binding Affinity of the Anti-PD-1 Antibodies

Table 4-1 below shows in vitro binding affinity data of exemplaryantibody Hu12A11.2b4 in comparison to literature anti-PD-1 antibodynivolumab prepared according to the procedures found in U.S. Pat. No.9,073,994. Hu12A11.2b4 exhibited similar binding properties to PD-1 ascompared to nivolumab according to surface plasmon resonance, ELISAassay with human (Hu) or cynomolgus (Cyno) PD-1, or in human Jurkatcells as measured in the assays of Example 1.

TABLE 4-1 Binding Properties of Select Antibodies against PD-1 Hu ELISAHu ELISA Cyno Hu Jurkat SPR EC₅₀ OD₆₅₀ ELISA EC₅₀ Antibody K_(D) (M)*(pM) max EC₅₀ (pM) (pM) Nivolumab 2.9E−09 101 2.2 70 56 Hu12A11.2b42.1E−09 197 2.9 102 45 *SPR = surface plasmon resonance as determinedaccording to Example 1; exponential notation shown (e.g., 3.0E−09 = 3.0× 10⁻⁹).

Example 5: Biological Activity of Anti-PD-1 Antibody Hu12A11.2b4

Hu12A11.2b4 was evaluated for biological activity in a number of invitro human cell assays described in Example 1. As shown in Table 5-1,Hu12A11.2b4 demonstrated 180 pM binding to PD-1 in CD4+ T cells. Inaddition, the anti-PD-1 activity of Hu12A11.2b4 was shown to be mediatedat least in part by its ability to block PD-L1 or PD-L2 interaction withPD-1 as assessed by flow cytometry. This biological activity wasconsistent with the activity of Hu12A11.2b4 in recombinant Jurkat Tcells expressing firefly luciferase gene under the control of NFATresponse elements with constitutive expression of human PD-1 (JurkatNFAT assay).

TABLE 5-1 Biological Activity of Exemplary anti-PD-1 Antibodies CD4+T-cell PD-L1 PD-L2 binding blocking blocking Jurkat NFAT Antibody EC₅₀(pM) (μg/mL) (μg/mL) EC₅₀ (μg/mL) Nivolumab 180 0.106 0.233 15 ± 20Hu12A11.2b4 180 0.045 0.132 4.5 ± 6.5

Anti-PD-1 antibody Hu12A11.2b4 further demonstrated an enhancement inimmunological response in in vitro assays. As shown in FIG. 4A,treatment with 10 μg/mL of Hu12A11.2b4 in mixed leukocyte cultureseffected a significant increase in IL-2 as well as an IFN-γ increase ofabout 7.4-fold. FIG. 4B shows that Hu12A11.2b4 exhibited a tetanustoxoid recall response of about 6-fold over no antibody treatment at 10μg/mL, with an EC₅₀=161 ng/mL.

The in vitro biological activity observed for Hu12A11.2b4 was similar tothat measured for the nivolumab used in Example 4. With respect toimmunological response, the comparison antibody nivolumab exhibited anIFN-γ increase of about 5.6-fold in MLR, and a tetanus toxoid recallresponse of about 6-fold over no antibody treatment at 10 μg/mL, with anEC₅₀=218 ng/mL.

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

What is claimed is:
 1. An anti-PD-1 binding protein which comprises (i)a V_(H) chain comprising three CDRs; and (ii) a V_(L) chain comprisingthree CDRs, wherein: V_(H) CDR#1 is (SEQ ID NO: 11) GYTFTHYGMN;V_(H) CDR#2 is (SEQ ID NO: 12) WVNTYTGEPTYADDFKG; V_(H) CDR#3 is(SEQ ID NO: 13) EGEGLGFGD; V_(L) CDR#1 is (SEQ ID NO: 14)RSSQSIVHSHGDTYLE; V_(L) CDR#2 is (SEQ ID NO: 15) KVSNRFS; andV_(L) CDR#3 is (SEQ ID NO: 16) FQGSHIPVT.


2. The anti-PD-1 binding protein of claim 1, which is humanized.
 3. Theanti-PD-1 binding protein of claim 2, which comprises a V_(H) chaincorresponding in sequence to SEQ ID NO:36; and a V_(L) chaincorresponding in sequence to SEQ ID NO:42.
 4. The anti-PD-1 bindingprotein of claim 3, which is an IgG.
 5. The anti-PD-1 binding protein ofclaim 4, which is an IgG₁.
 6. The anti-PD-1 binding protein of claim 5comprising a variant CH2 domain having amino acid substitutions L234Aand L235A.
 7. The anti-PD-1 binding protein of claim 4, which is anIgG₄.
 8. The anti-PD-1 binding protein of claim 7 comprising a variantFc region having an amino acid substitution S228P.
 9. The anti-PD-1binding protein of claim 3 comprising a kappa light constant region. 10.The anti-PD-1 binding protein of claim 3 which comprises a heavy chaincorresponding in sequence to SEQ ID NO:51 or SEQ ID NO:52, and a lightchain corresponding in sequence to SEQ ID NO:61.
 11. The anti-PD-1binding protein of claim 1 which has a K_(D) of less than about 100 nM.12. The anti-PD-1 binding protein of claim 11 which has a K_(D) of lessthan about 10 nM.
 13. A pharmaceutical composition comprising theanti-PD-1 binding protein of claim 1, and a pharmaceutically acceptablecarrier.
 14. A nucleic acid comprising a nucleotide sequence encoding ananti-PD-1 binding protein, wherein the binding protein comprises (i) aV_(H) chain comprising three CDRs; and (ii) a V_(L) chain comprisingthree CDRs, wherein: V_(H) CDR#1 is (SEQ ID NO: 11) GYTFTHYGMN;V_(H) CDR#2 is (SEQ ID NO: 12) WVNTYTGEPTYADDFKG; V_(H) CDR#3 is(SEQ ID NO: 13) EGEGLGFGD; V_(L) CDR#1 is (SEQ ID NO: 14)RSSQSIVHSHGDTYLE; V_(L) CDR#2 is (SEQ ID NO: 15) KVSNRFS; andV_(L) CDR#3 is (SEQ ID NO: 16) FQGSHIPVT.


15. A vector comprising the nucleic acid of claim
 14. 16. A prokaryotichost cell transformed with the vector of claim
 15. 17. A eukaryotic hostcell transformed with the vector of claim
 15. 18. A eukaryotic host cellengineered to express the nucleic acid of claim
 14. 19. The eukaryotichost cell of claim 18 which is a mammalian host cell.
 20. A method ofproducing an anti-PD-1 binding protein thereof, comprising: (a)culturing the host cell of claim 19 and (b) recovering the anti-PD-1binding protein.
 21. A method of activating the immune system,comprising administering to a patient in need thereof an effectiveamount of an anti-PD-1 binding protein expressed by the host cell ofclaim
 18. 22. A method of treating a cancer, comprising administering toa patient in need thereof a therapeutically effective amount of ananti-PD-1 binding protein, wherein the binding protein comprises (i) aV_(H) chain comprising three CDRs; and (ii) a V_(L) chain comprisingthree CDRs, wherein: V_(H) CDR#1 is (SEQ ID NO: 11) GYTFTHYGMN;V_(H) CDR#2 is (SEQ ID NO: 12) WVNTYTGEPTYADDFKG; V_(H) CDR#3 is(SEQ ID NO: 13) EGEGLGFGD; V_(L) CDR#1 is (SEQ ID NO: 14)RSSQSIVHSHGDTYLE; V_(L) CDR#2 is (SEQ ID NO: 15) KVSNRFS; andV_(L) CDR#3 is (SEQ ID NO: 16) FQGSHIPVT.


23. The method of claim 22, wherein the cancer is selected from bladdercancer, breast cancer, head and neck cancer, kidney cancer, lung cancer,lymphoma, melanoma, and stomach cancer.
 24. The method of claim 23,wherein the lung cancer is non-small cell lung cancer.
 25. The method ofclaim 21, in which the anti-PD-1 binding protein is administeredintravenously.